Opsin-Binding Ligands, Compositions and Methods of Use

ABSTRACT

Compounds and compositions of said compounds along with methods of use of compounds are disclosed for treating ophthalmic conditions related to mislocalization of opsin proteins, the misfolding of mutant opsin proteins and the production of toxic visual cycle products that accumulate in the eye. Compounds and compositions useful in the these methods, either alone or in combination with other therapeutic agents, are also described.

PRIORITY CLAIM

This application claims priority of U.S. Provisional Application61/268,757, filed 16 Jun. 2009, the disclosure of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to compounds and compositions thereof foruse in the treatment and/or prevention of ophthalmic diseases.

BACKGROUND OF THE INVENTION

A diminished visual acuity or total loss of vision may result from anumber of eye diseases or disorders caused by dysfunction of tissues orstructures in the anterior segment of the eye and/or posterior segmentof the eye. Of those that occur as a consequence of a dysfunction in theanterior segment, aberrations in the visual cycle are often involved.The visual cycle (also frequently referred to as the retinoid cycle)comprises a series of light-driven and/or enzyme catalyzed reactionswhereby a light-sensitive chromophore (called rhodopsin) is formed bycovalent bonding between the protein opsin and the retinoid agent11-cis-retinal and subsequently, upon exposure to light, the11-cis-retinal is converted to all-trans-retinal, which can then beregenerated into 11-cis-retinal to again interact with opsin. A numberof visual, ophthalmic, problems can arise due to interference with thiscycle. It is now understood that at least some of these problems are dueto improper protein folding, such as that of the protein opsin.

The main light and dark photoreceptor in the mammalian eye is the rodcell, which contains a folded membrane containing protein molecules thatcan be sensitive to light, the main one being opsin. Like other proteinspresent in mammalian cells, opsin is synthesized in the endoplasmicreticulum (i.e., on ribosomes) of the cytoplasm and then conducted tothe cell membrane of rod cells. In some cases, such as due to geneticdefects and mutation of the opsin protein, opsin can exhibit improperfolding to form a conformation that either fails to properly insert intothe membrane of the rod cell or else inserts but then fails to properlyreact with 11-cis-retinal to form native rhodopsin. In either case, theresult is moderate to severe interference with visual perception in theanimal so afflicted.

Among the diseases and conditions linked to improper opsin folding isretinitis pigmentosa (RP), a progressive ocular-neurodegenerativedisease (or group of diseases) that affects an estimated 1 to 2 millionpeople worldwide. In RP, photoreceptor cells in the retina are damagedor destroyed, leading to loss of peripheral vision (i.e., tunnel vision)and subsequent partial or near-total blindness.

In the American population the most common defect occurs as a result ofreplacement of a proline residue by a histidine residue at amino acidnumber 23 in the opsin polypeptide chain (dubbed “P23H”), caused by amutation in the gene for opsin. The result is production of adestabilized form of the protein, which is misfolded and aggregates inthe cytoplasm rather than being transported to the cell surface. Likemany other protein conformational diseases (PCD₆), the clinically commonP23H opsin mutant associated with autosomal dominant RP is misfolded andretained intracellularly. The aggregation of the misfolded protein isbelieved to result in photoreceptor damage and cell death.

Recent studies have identified small molecules that stabilize misfoldedmutant proteins associated with disease. Some of these, dubbed “chemicalchaperones,” stabilize proteins non-specifically. Examples of theseinclude glycerol and trimethylamine oxide. These are not very desirablefor treating ophthalmic disease because such treatment usually requireshigh dosages that may cause toxic side effects. Other agents, dubbed“pharmacological chaperones,” (which include native ligands andsubstrate analogs) act to stabilize the protein by binding to specificsites and have been identified for many misfolded proteins, e.g.,G-protein coupled receptors. Opsin is an example of a G-protein coupledreceptor and its canonical pharmacological chaperones include the classof compounds referred to as retinoids. Thus, certain retinoid compoundshave been shown to stabilize mutant opsin proteins (see, for example,U.S. Patent Pub. 2004-0242704, as well as Noorwez et al., J. Biol.Chem., 279(16): 16278-16284 (2004)).

The visual cycle comprises a series of enzyme catalyzed reactions,usually initiated by a light impulse, whereby the visual chromophore ofrhodopsin, consisting of opsin protein bound covalently to11-cis-retinal, is converted to an all-trans-isomer that is subsequentlyreleased from the activated rhodopsin to form opsin and theall-trans-retinal product. This part of the visual cycle occurs in theouter portion of the rod cells of the retina of the eye. Subsequentparts of the cycle occur in the retinal pigmented epithelium (RPE).Components of this cycle include various enzymes, such as dehydrogenasesand isomerases, as well as transport proteins for conveying materialsbetween the RPE and the rod cells.

As a result of the visual cycle, various products are produced, calledvisual cycle products. One of these is all-trans-retinal produced in therod cells as a direct result of light impulses contacting the11-cis-retinal moiety of rhodopsin. All-trans-retinal, after releasefrom the activated rhodopsin, can be regenerated back into11-cis-retinal or can react with an additional molecule ofall-trans-retinal and a molecule of phosphatidylethanolamine to produceN-retinylidene-N-retinylethanolamine (dubbed “A2E”), an orange-emittingfluorophore that can subsequently collect in the rod cells and in theretina pigmented epithelium (RPE). As A2E builds up (as a normalconsequence of the visual cycle) it can also be converted intolipofuscin, a toxic substance that has been implicated in severalabnormalities, including ophthalmic conditions such as wet and dry agerelated macular degeneration (ARMD). A2E can also prove toxic to the RPEand has been associated with dry ARMD.

Because the build-up of toxic visual cycle products is a normal part ofthe physiological process, it is likely that all mammals, especially allhumans, possess such an accumulation to some extent throughout life.However, during surgical procedures on the eye, especially on theretina, where strong light is required over an extended period, forexample, near the end of cataract surgery and while implanting the newlens, these otherwise natural processes can cause toxicity because ofthe build-up of natural products of the visual cycle. Additionally,excessive rhodopsin activation as a result of bright light stimulationcan cause photoreceptor cell apoptosis via an AP-1 transcription factordependent mechanism. Because of this, there is a need for agents thatcan be administered prior to, during or after (or any combination ofthese) the surgical process and that has the effect of inhibitingrhodopsin activation as well as reducing the production of visual cycleproducts that would otherwise accumulate and result in toxicity to theeye, especially to the retina.

The present invention answers this need by providing small moleculeswhich noncovalently bind to opsin or mutated forms of opsin for treatingand/or amelioration such conditions, if not preventing them completely.Importantly, such agents are not natural retinoids and thus are nottightly controlled for entrance into the rod cells, where mutated formsof opsin are synthesized and/or visual cycle products otherwiseaccumulate. Therefore, such agents can essentially be titrated in asneeded for facilitating the proper folding trafficking of mutated opsinsto the cell membrane or prevention of rhodopsin activation that can leadto the excessive build-up of visual cycle products likeall-trans-retinal that in turn can lead to toxic metabolic products.Such compounds may compete with 11-cis-retinal to reduceall-trans-retinal by tying up the retinal binding pocket of opsin toprevent excessive all-trans-retinal build up. Thus, the compoundsprovided by the present invention have the advantage that they do notdirectly inhibit the enzymatic processes by which 11-cis-retinal isproduced in the eye (thus not contributing to retinal degeneration).Instead, the formation of all-trans-retinal is limited and thereby theformation of A2E is reduced. Finally, by limiting the ability of11-cis-retinal to combine with opsin to form rhodopsin, rhodopsinactivation caused by bright light stimulation especially duringophthalmic surgery is also diminished thus preventing the photocelldeath that results.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Inboth cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision. The present invention solves this problem by providing amethod of correcting mislocalized opsin within a photoreceptor cell bycontacting a mislocalized opsin protein with an opsin-binding agent thatbinds reversibly and/or non-covalently to said mislocalized opsinprotein, and promotes the appropriate intracellular processing andtransport of said opsin protein. This correction of mislocalizationrelieves photoreceptor cell stress, preventing decline in viability anddeath of photoreceptor cells in various diseases of vision loss, and innormal age-related decline in dim-light and peripheral rod-mediatedvision, central cone-mediated vision, and loss of night vision.

Computer-assisted molecular docking has lead to the successful discoveryof novel ligands for more than 30 targets (Shoichet et al., Curr. Opin.in Chem. Biol. 6: 439-46 (2002)). This strategy has been appliedprimarily to enzymes, such as aidose reductase (Iwata et al., J. Med.Chem. 44: 1718-28 (2001)), Bcl-2, matriptase (Enyedy et al., J. Med.Chem. 44: 1349-55 (2001)), adenovirus protease (Pang et al., FEBSLetters 502: 93-97 (2001)), AmpC fl-lactamase, carbonic anhydrase(Gruneberg et al., J. Med. Chem. 45: 3588-602 (2002)), HPRTase (Freymannet al., Chemistry & Biology 7: 957-68 (2000)), dihydrodipicolinate(Paiva et al., Biochimica Biophysica Acta 1545: 67-77 (2001)) and Cdk4(Honma et al., J. Med. Chem. 44: 4615-27 (2001)). Improvements indocking algorithms and multiprocessor resources have improved thetechnique of computer-assisted molecular docking such that it can now beapplied to more challenging problems. For example, this approach hasrecently been applied to defining small molecules that targetprotein-protein interfaces, which are relatively broad and flat comparedto easily targeted enzyme active sites.

More recently, a new computational technique defining the thermodynamicproperties and phase behavior of water in confined regions of proteinpockets has been developed (Young et. al., PNAS 104: 808-13 (2007)). Thealgorithm developed has been utilized to characterize the solvation ofprotein pockets. The molecular dynamics simulations and solvent analysistechniques have characterized the solvation of hydrophobic enclosuresand correlated hydrogen bonds as inducing atypical entropic andenthalpic penalties of hydration which stabilize the protein-ligandcomplex with respect to the independently solvated ligand and protein.These criteria, commonly referred to as the water map, have been used torationalize Factor Xa ligand binding (Abel et. al., JACS 130: 2817-31(2008)).

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds having thestructure of Formula I, including pharmaceutically acceptable salts,solvates and hydrates thereof, and compositions of said compounds:

A-B-Q-V   Formula I

wherein A, B, Q, and V are as described elsewhere herein.

In another aspect, the present invention provides compounds having thestructure of Formula II,

wherein A, B, Q, W and p are as described elsewhere herein, includingpharmaceutically acceptable salts, solvates and hydrates thereof, andcompositions of said compounds.

In a related aspect, the present invention relates to a method ofinhibiting the formation or accumulation of a visual cycle product,comprising contacting an opsin protein with a compound recited herein toinhibit formation of said visual cycle product relative to when saidcontacting does not occur.

In a further aspect, the present invention relates to a method to reducethe light toxicity associated with ophthalmic surgery by preventingrhodopsin regeneration during surgery to a mammalian eye and/or preventor slow the formation of toxic visual cycle products by fractionallypreventing rhodopsin formation during periods of light activationthereby providing a treatment of ocular conditions associated with thebuild up of visual products such as wet or dry ARMD.

In yet a further aspect, the present invention relates to a method ofcorrecting the proper folding and trafficking of mutated opsin proteins,comprising contacting a mutated opsin protein with a compound thatstabilizes the proper three dimensional conformation of the proteinrelative to when said contacting does not occur wherein the compound hasthe structure of Formula I and/or Formula II including pharmaceuticallyacceptable salts, solvates and hydrates thereof.

In one embodiment, the ligand selectively binds reversibly ornon-covalently to opsin. In another embodiment, the ligand binds at ornear the 11-cis-retinal binding pocket of the opsin protein. In yetanother embodiment, the ligand binds to the opsin protein so as toinhibit or slow the covalent binding of 11-cis-retinal to the opsinprotein when the 11-cis-retinal is contacted with the opsin protein inthe presence of the ligand. In yet another embodiment, the ligand bindsto the opsin in the retinal binding pocket of opsin protein or disrupts11-cis-retinal binding to the retinal binding pocket of opsin. In yetanother embodiment, the ligand binds to the opsin protein so as toinhibit covalent binding of 11-cis-retinal to the opsin protein. In yetanother embodiment, the mammal is a human being.

In yet another embodiment, slowing or halting the progression of wet ordry ARMD is associated with reducing the level of a visual cycleproduct, for example, a visual cycle product formed fromall-trans-retinal, such as lipofuscin orN-retinylidine-N-retinylethanolamine (A2E). In yet another embodimentslowing or halting the progression of RP is associated with correctingthe folding of mutated opsins. In another embodiment, the administeringis topical administration, local administration (e.g., intraocular orperiocular injection or implanti) or systemic administration (e.g.,oral, injection). In yet another embodiment, the light toxicity isrelated to an ophthalmic procedure (e.g., ophthalmic surgery). In stillanother embodiment, the administering occurs prior to, during, or afterthe ophthalmic surgery.

In one aspect, the invention provides a method of correctingmislocalized opsin within a photoreceptor cell, comprising contacting amislocalized opsin protein with an opsin-binding agent that bindsreversibly and/or non-covalently to said mislocalized opsin protein topromote the appropriate intracellular processing and transport of saidopsin protein.

In various embodiments, the ophthalmic condition is any one or more ofwet or dry form of macular degeneration, retinitis pigmentosa, a retinalor macular dystrophy, Stargardt's disease, Sorsby's dystrophy, autosomaldominant drusen, Best's dystrophy, periphenn mutation associate withmacular dystrophy, dominant form of Stargart's disease, North Carolinamacular dystrophy, light toxicity, retinitis pigmentosa, normal visionloss related aging and normal loss of night vision related to aging.

In still another embodiment, the method further involves administeringto a mammal, preferably a human being, an effective amount of at leastone additional agent selected from the group consisting of a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp90 chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, anda histone deacetylase inhibitor. In yet another embodiment, the opsinbinding ligand and the additional agent are administered simultaneously.

In still another embodiment, the opsin binding ligand and the additionalagent are each incorporated into a composition that provides for theirlong-term release. In another embodiment, the composition is part of amicrosphere, nanosphere, nano emulsion or implant. In anotherembodiment, the composition further involves administering a mineralsupplement, at least one anti-inflammatory agent, such as a steroid(e.g., any one or more of cortisone, hydrocortisone, prednisone,prednisolone, methylprednisolone, triamcinolone, betamethasone,beclamethasone and dexamethasone), or at least one anti-oxidant, such asvitamin A, vitamin C and vitamin E. In various embodiments, the opsinbinding ligand, the anti-inflammatory agent, and/or the anti-oxidant areadministered simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows predicted hydration of the rod opsin retinal binding pocketas developed from a homology model of human rhodopsin based upon thecrystal structure of bovine rhodopsin. As a reference, the surfacevolume of 11-cis retinal is indicated by general outline and thestructure of 11-cis retinal is indicated by bold black lines. Specifichydration sites are shown as circles where water molecules would bepredicted to reside within the pocket in the absence of a ligand.Circles labeled with a “D” designate hydration sites that are in veryhydrophobic environments and thus upon displacement by a ligand arepredicted to lower the energy of the ligand protein complex relative tothe hydrated apoprotein. Circles labeled with a “R” designate hydrationsites where the water molecule is forming stable hydrogen bonds withfunctional groups on the protein and thus signify coordinates within thebinding pocket where suitable hydrogen bonding functionality of theligand should be incorporated to replace the hydrogen bondinginteractions that are broken between the water molecule and the proteinupon binding of the ligand.

FIG. 2 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 6 during mutant protein production relative to pigmentformation in the presence of vehicle, here dimethylsulfoxide (DMSO),alone.

FIG. 3 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 13 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 4 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 33 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 5 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 37 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 6 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 50 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 7 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 51 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 8 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 52 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 9 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 53 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 10 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 55 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 11 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 57 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 12 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 63 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 13 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 71 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 14 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 73 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 15 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 80 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 16 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 105 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

FIG. 17 shows the increase in regeneration of 500 nm absorbing pigmentupon treatment with retinal from P23H opsin that was treated with 20 μMof compound 106 during mutant protein production relative to pigmentformation in the presence of vehicle (DMSO) alone.

DEFINITIONS

As used throughout the disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings.

By “mislocalization” of a photoreceptor cell visual pigment protein (forexample, opsin, especially human opsin) is meant that the synthesizedprotein is not found at the normal or appropriate cellular location.

“Pharmacologic chaperones” refer to small molecular weight chemicalcompounds that interact with a protein (usually with a mis-folded, orun-folded protein) in such a way as to alter the folding or confirmationof said protein. Such an interaction can have diverse consequences onthe cellular fate of the protein, including but not limited to leadingto increased stability and increased levels of functional protein,increased stability and increased levels of non-functional protein, ordecreased stability and decreased levels of functional or non-functionalprotein.

“Productive chaperone” refers to a pharmacologic chaperone that wheninteracting with a protein leads to an increased level of functionalprotein.

“Counterproductive, shipwreck or destructive chaperone” refers to apharmacologic chaperone that interacts with a protein (usually with amis-folded, or un-folded protein) and this interaction leads to adecreased stability and/or decreased levels of functional ornon-functional protein.

By “proteasomal inhibitor” is meant a compound that reduces aproteasomal activity, such as the degradation of a ubiquinated protein.

By “autophagy inhibitor” is meant a compound that reduces thedegradation of a cellular component by a cell in which the component islocated.

By “lysosomal inhibitor” is meant a compound that reduces theintracellular digestion of macromolecules by a lysosome. In oneembodiment, a lysosomal inhibitor decreases the proteolytic activity ofa lysosome.

By “Inhibitor of ER-Golgi protein transport” is meant a compound thatreduces the transport of a protein from the ER (endoplasmic reticulum)to the Golgi, or from the Golgi to the ER.

By “HSP90 chaperone inhibitor” is meant a compound that reduces thechaperone activity of heat shock protein 90 (HSP90). In one embodiment,the inhibitor alters protein binding to an HSP90 ATP/ADP pocket.

By “heat shock response activator” is meant a compound that increasesthe chaperone activity or expression of a heat shock pathway component.Heat shock pathway components include, but are not limited to, HSP100,HSP90, HSP70, HASP60, HSP40 and small HSP family members.

By “glycosidase inhibitor” is meant a compound that reduces the activityof an enzyme that cleaves a glycosidic bond.

By “histone deacetylase inhibitor” is meant a compound that reduces theactivity of an enzyme that deacetylates a histone.

By “reduces” or “increases” is meant a negative or positive alteration,respectively. In particular embodiments, the alteration is by at leastabout 10%, 25%, 50%, 75%, or 100% of the initial level of the proteinproduced in the absence of the opsin binding ligand.

As used herein, the term “wild-type conformation” refers to the threedimensional conformation or shape of a protein that is free of mutationsto its amino acid sequence. For opsin, this means a protein free frommutations that cause misfiling, such as the mutation designated P23H(meaning that a proline is replaced by a histidine at residue 23starting from the N-terminus). Opsin in a “wild-type conformation” iscapable of opsin biological function, including but not limited to,retinoid binding, visual cycle function, and insertion into aphotoreceptor membrane.

By “agent” is meant a small compound (also called a “compound”),polypeptide, polynucleotide, or fragment thereof. The terms compound andagent are used interchangeably unless specifically stated otherwiseherein for a particular agent or compound.

By “correcting the conformation” of a protein is meant inducing theprotein to assume a conformation having at least one biological activityassociated with a wild-type protein.

By “misfolded opsin protein” is meant a protein whose tertiary structurediffers from the conformation of a wild-type protein, such that themisfolded protein lacks one or more biological activities associatedwith the wild-type protein.

By “selectively binds” is meant a compound that recognizes and binds apolypeptide of the invention, such as opsin, but which does notsubstantially recognize and bind other molecules, especially non-opsinpolypeptides, in a sample, for example, a biological sample.

By “effective amount” or “therapeutically effective amount” is meant alevel of an agent sufficient to exert a physiological effect on a cell,tissue, or organ or a patient. As used herein, it is the amountsufficient to effect the methods of the invention to achieve the desiredresult.

By “pharmacological chaperone” is meant a molecule that upon contactinga mutant protein is able to facilitate/stabilize the proper folding ofthe protein such that it acts and functions much more like wild typeprotein than would be the case in the absence of the molecule.

By “control” is meant a reference condition. For example, where a cellcontacted with an agent of the invention is compared to a correspondingcell not contacted with the agent, the latter is the “control” or“control” cell.

By “treat” is meant decrease, suppress, attenuate, diminish, arrest, orstabilize the development or progression of a disease, preferably anocular disease, such as RP, AMD and/or light toxicity.

By “prevent” is meant reduce the risk that a subject will develop acondition, disease, or disorder, preferably an ocular disease, such asRP, AMD and/or light toxicity.

By “competes for binding” is meant that a compound of the invention andan endogenous ligand are incapable of binding to a target at the sametime. Assays to measure competitive binding are known in the art, andinclude, measuring a dose dependent inhibition in binding of a compoundof the invention and an endogenous ligand by measuring t_(1/2), forexample.

A “pharmaceutically acceptable salt” is a salt formed from an acid or abasic group of one of the compounds of the invention. Illustrative saltsinclude, but are not limited to, sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbatc, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesuifonate, and pamoate (i.e.,1,1′-methytene-bis-(2-hydroxy-3-naphthoate)) salts.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound of the invention having an acidic functionalgroup, such as a carboxylic acid functional group, and apharmaceutically acceptable inorganic or organic base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine;tributyl amine; pyridine; N-methyl-N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-hydroxy-lower alkylamines), suchas mono-, bis-, or tris-(2-hydroxyethyl)-amine,2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such asN,N-dimethyl-N-(2-hydroxyethyl)-amine, or tri-(2-hydroxyethyl)amine;N-methyl-D-glucamine; and amino acids such as arginine, lysine, and thelike.

The term “pharmaceutically acceptable salt” also refers to a saltprepared from a compound disclosed herein, e.g., a salt of a compound ofExample 1, having a basic functional group, such as an amino functionalgroup, and a pharmaceutically acceptable inorganic or organic acid.Suitable acids include, but are not limited to, hydrogen sulfate, citricacid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide,hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lacticacid, salicylic acid, tartaric acid, ascorbic acid, succinic acid,maleic acid, besylic acid, fumaric acid, gluconic acid, glucaronic acid,saccharic acid, formic acid, benzoic acid, glutamic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, andp-toluenesulfonic acid.

The term “pharmaceutically-acceptable excipient” as used herein meansone or more compatible solid or liquid tiller, diluents or encapsulatingsubstances that are suitable for administration into a human. The term“excipient” includes an inert substance added to a pharmacologicalcomposition to further facilitate administration of a compound. Examplesof excipients include but are not limited to calcium carbonate, calciumphosphate, various sugars and types of starch, cellulose derivatives,gelatin, vegetable oils and polyethylene glycols.

The term “carrier” denotes an organic or inorganic ingredient, naturalor synthetic, with which the active ingredient is combined to facilitateadministration.

The term “parenteral” includes subcutaneous, intrathecal, intravenous,intramuscular, intraperitoncal, or infusion.

The term “visual cycle product” refers to a chemical entity produced asa natural product of one or more reactions of the visual cycle (thereactive cycle whereby opsin protein binds 11-cis-retinal to formrhodopsin, which accepts a light impulse to convert 11-cis-retinal toall trans-retinal, which is then released from the molecule toregenerate opsin protein with subsequent binding of a new 11-cis-retinalto regenerate rhodopsin). Such visual cycle products include, but arenot limited to, all-trans-retinal, lipofuscin and A2E.

The term “light toxicity” refers to any condition affecting vision thatis associated with, related to, or caused by the production and/oraccumulation of visual cycle products. Visual cycle products include,but are not limited to, all-trans-retinal, lipofuscin or A2E. In oneparticular embodiment, light toxicity is related to exposure of the eyeto large amounts of light or to very high light intensity, occurring,for example, during a surgical procedure on the retina.

The term “opsin” refers to an opsin protein, preferably a mammalianopsin protein, most preferably a human opsin protein. In one embodiment,the opsin protein is in the wild-type (i.e., physiologically active)conformation. One method of assaying for physiological activity isassaying the ability of opsin to bind 11-cis-retinal and form activerhodopsin. A mutant opsin, such as the P23H mutant, that is ordinarilymisfolded has a reduced ability to bind 11-cis-retinal, and thereforeforms little or no rhodopsin. Where the conformation of the mutant opsinhas been corrected (for example, by binding to a pharmacologicalchaperone), the opsin is correctly inserted into the rod cell membraneso that its conformation is the same, or substantially the same, as thatof a non-mutant opsin. This allows the mutant opsin to bind11-cis-retinal to form active rhodopsin. Therefore, the methods of theinvention operate to reduce the formation of visual cycle products.

“Alkyl” refers to an unbroken non-cyclic chain of carbon atoms that maybe substituted with other chemical groups. It may also be branched orunbranched, substituted or unsubstituted.

“Lower alkyl” refers to a branched or straight chain acyclic alkyl groupcomprising one to ten carbon atoms, preferably one to eight carbonatoms, more preferably one to six carbon atoms. Exemplary lower alkylgroups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, pentyl, neopentyl, iso-amyl, hexyl, and octyl.

All alkyl, alkenyl or alkynyl groups disclosed herein may be substitutedwith one or more of the following: lower alkyl, hydroxy, ester, amidyl,oxo, carboxyl, carboxamido, halo, cyano, nitrate, nitrite, thionitrate,thionitrite sulfhydryl and amino groups (as elsewhere defined herein).

“Haloalkyl” refers to a lower alkyl group, an alkenyl group, an alkynylgroup, a bridged cycloalkyl group, a cycloalkyl group or a heterocyclicring, as defined herein, to which is appended one or more halogens, asdefined herein. Exemplary haloalkyl groups include trifluoromethyl,chloromethyl, 2-bromobutyl and 1-bromo-2-chloro-pentyl.

“Alkenyl” refers to a branched or straight chain C₂-C₁₀ hydrocarbon(preferably a C₂-C₈ hydrocarbon, more preferably a C₂-C₆ hydrocarbon)that can comprise one or more carbon-carbon double bonds. Exemplaryalkenyl groups include propylenyl, buten-1-yl, isobutenyl, penten-1-yl,2,2-methylbuten-1-yl, 3-methylbuten-1-yl, hexan-1-yl, hepten-1-yl andocten-1-yl.

“Lower alkenyl” refers to a branched or straight chain C₂-C₄ hydrocarbonthat can comprise one or two carbon-carbon double bonds.

“Substituted alkenyl” refers to a branched or straight chain C₂-C₁₀hydrocarbon (preferably a C₂-C₈ hydrocarbon, more preferably a C₂-C₅hydrocarbon) which can comprise one or more carbon-carbon double bonds,wherein one or more of the hydrogen atoms have been replaced with one ormore R¹⁰⁰ groups, wherein each R¹⁰⁰ is independently a hydroxy, an oxo,a carboxyl, a carboxamido, a halo, a cyano or an amino group, as definedherein.

“Alkynyl” refers to an unsaturated acyclic C₂-C₁₀ hydrocarbon(preferably a C₂-C₈ hydrocarbon, more preferably a C₂-C₆ hydrocarbon)that can comprise one or more carbon-carbon triple bonds. Exemplaryalkynyl groups include ethynyl, propynyl, butyn-1-yl, butyn-2-yl,pentyl-1-yl, pentyl-2-yl, 3-methylbutyn-1-yl, hexyl-1-yl, hexyl-2-yl,hexyl-3-yl and 3,3-dimethyl-butyn-1-yl.

“Lower alkynyl” refers to a branched or straight chain C₂-C₄ hydrocarbonthat can comprise one or two carbon-carbon triple bonds

“Bridged cycloalkyl” refers to two or more cycloalkyl groups,heterocyclic groups, or a combination thereof fused via adjacent ornon-adjacent atoms. Bridged cycloalkyl groups can be unsubstituted orsubstituted with one, two or three substituents independently selectedfrom alkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, halo,carboxyl, alkylcarboxylic acid, aryl, amidyl, ester, alkylcarboxylicester, carboxamido, alkylcarboxamido, oxo and nitro. Exemplary bridgedcycloalkyl groups include adamantyl, decahydronapthyl, quinuclidyl,2,6-dioxabicyclo(3.3.0)octane, 7-oxabicyclo(2.2.1)heptyl and8-azabicyclo(3,2,1)oct-2-enyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarboncomprising from about 3 to about 10 carbon atoms. Cycloalkyl groups canbe unsubstituted or substituted with one, two or three substituentsindependently selected from alkyl, alkoxy, amino, alkylamino,dialkylamino, arylamino, diarylamino, alkylarylamino, aryl, amidyl,ester, hydroxy, halo, carboxyl, alkylcarboxylic acid, alkylcarboxylicester, carboxamido, alkylcarboxamido, oxo, alkylsulfinyl, and nitro.Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclohexenyl and cyclohepta-1,3-dienyl.

“Heterocyclic ring or group” refers to a saturated or unsaturated cyclicor polycyclic hydrocarbon group having about 2 to about 12 carbon atomswhere 1 to about 4 carbon atoms are replaced by one or more nitrogen,oxygen and/or sulfur atoms. Sulfur may be in the thio, sulfinyl orsulfonyl oxidation state. The heterocyclic ring or group can be fused toan aromatic hydrocarbon group. Heterocyclic groups can be unsubstitutedor substituted with one, two or three substituents independentlyselected from alkyl, alkoxy, amino, alkylthio, aryloxy, arylthio,arylalkyl, hydroxy, oxo, thial, halo, carboxyl, carboxylic ester,alkylcarboxylic acid, alkylcarboxylic ester, aryl, arylcarboxylic acid,arylcarboxylic ester, amidyl, ester, alkylcarbonyl, arylcarbonyl,alkylsulfinyl, carboxamido, alkylcarboxamido, arylcarboxamido, sulfonicacid, sulfonic ester, sulfonamide nitrate and nitro. Exemplaryheterocyclic groups include pyrrolyl, furyl, thienyl, 3-pyrrolinyl,4,5,6-trihydro-2H-pyranyl, pyrdinyl, 1,4-dihydropyridinyl, pyrazolyl,triazolyl, pyrimidinyl, pyridazinyl, oxazolyl, thiazolyl,thieno[2,3-d]pyrimidine, 4,5,6,7-tetrahydrobenzo[b]thiophene,imidazolyl, indolyl, thiophenyl, furanyl, tetrahydrofuranyl, tetrazolyl,pyrrolinyl, pyrrolindinyl, oxazolindinyl 1,3-dioxolanyl, imidazolinyl,imidazolindinyl, pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl,1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 2H-pyranyl,4H-pyranyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl,thiomorpholinyl, pyrazinyl, piperazinyl, 1,3,5-triazinyl,1,3,5-trithianyl, benzo(b)thiophenyl, benzimidazolyl, benzothiazolinyl,quinolinyl and 2,6-dioxabicyclo(3.3.0)octane.

“Heterocyclic compounds” refer to mono- and polycyclic compoundscomprising at least one aryl or heterocyclic ring.

“Aryl” refers to a monocyclic, bicyclic, carbocyclic or heterocyclicring system comprising one or two aromatic rings. Exemplary aryl groupsinclude phenyl, pyridyl, napthyl, quinoyl, tetrahydronaphthyl, furanyl,indanyl, indenyl, indoyl. Aryl groups (including bicyclic aryl groups)can be unsubstituted or substituted with one, two or three substituentsindependently selected from alkyl, alkoxy, alkylthio, amino, alkylamino,dialkylamino, arylamino, diarylamino, alkylarylamino, halo, cyano,alkylsulfinyl, hydroxy, carboxyl, carboxylic ester, alkylcarboxylicacid, alkylcarboxylic ester, aryl, arylcarboxylic acid, arylcarboxylicester, alkylcarbonyl, arylcarbonyl, amidyl, ester, carboxamido,alkylcarboxamido, carbomyl, sulfonic acid, sulfonic ester, sulfonamidoand nitro. Exemplary substituted aryl groups include tetrafluorophenyl,pentafluorophenyl, sulfonamide, alkylsulfonyl and arylsulfonyl.

“Cycloalkenyl” refers to an unsaturated cyclic C₃-C₁₀ hydrocarbon(preferably a C₃-C₈ hydrocarbon, more preferably a C₃-C₆ hydrocarbon),which can comprise one or more carbon-carbon double bonds.

“Alkylaryl” refers to an alkyl group, as defined herein, to which isappended an aryl group, as defined herein. Exemplary alkylaryl groupsinclude benzyl, phenylethyl, hydroxybenzyl, fluorobenzyl andfluorophenylethyl.

“Arylalkyl” refers to an aryl radical, as defined herein, attached to analkyl radical, as defined herein. Exemplary arylalkyl groups includebenzyl, phenylethyl, 4-hydroxybenzyl, 3-fluorobenzyl and2-fluorophenylethyl.

“Arylalkenyl” refers to an aryl radical, as defined herein, attached toan alkenyl radical, as defined herein. Exemplary arylalkenyl groupsinclude styryl and propenylphenyl.

“Cycloalkylalkyl” refers to a cycloalkyl radical, as defined herein,attached to an alkyl radical, as defined herein.

“Cycloalkylalkoxy” refers to a cycloalkyl radical, as defined herein,attached to an alkoxy radical, as defined herein.

“Cycloalkylalkylthio” refers to a cycloalkyl radical, as defined herein,attached to an alkylthio radical, as defined herein.

“Heterocyclicalkyl” refers to a heterocyclic ring radical, as definedherein, attached to an alkyl radical, as defined herein.

“Arylheterocyclic ring” refers to a bi- or tricyclic ring comprised ofan aryl ring, as defined herein, appended via two adjacent carbon atomsof the aryl ring to a heterocyclic ring, as defined herein. Exemplaryarylheterocyclic rings include dihydroindole and1,2,3,4-tetra-hydroquinoline.

“Alkylheterocyclic ring” refers to a heterocyclic ring radical, asdefined herein, attached to an alkyl radical, as defined herein.Exemplary alkylheterocyclic rings include 2-pyridylmethyl and1-methylpiperidin-2-one-3-methyl.

“Alkoxy” refers to R₅₀O—, wherein R₅₀ is an alkyl group, as definedherein (preferably a lower alkyl group or a haloalkyl group, as definedherein). Exemplary alkoxy groups include methoxy, ethoxy, t-butoxy,cyclopentyloxy and trifluoromethoxy.

“Aryloxy” refers to R₅₅O—, wherein R₅₅ is an aryl group, as definedherein. Exemplary arylkoxy groups include napthyloxy, quinolyloxy,isoquinolizinyloxy.

“Alkylthio” refers to R₅₀S—, wherein R₅₀ is an alkyl group, as definedherein.

“Lower alkylthio” refers to a lower alkyl group, as defined herein,appended to a thio group, as defined herein.

“Arylalkoxy” or “alkoxyaryl” refers to an alkoxy group, as definedherein, to which is appended an aryl group, as defined herein. Exemplaryarylalkoxy groups include benzyloxy, phenylethoxy andchlorophenylethoxy.

“Arylalklythio” refers to an alkylthio group, as defined herein, towhich is appended an aryl group, as defined herein. Exemplaryarylalklythio groups include benzylthio, phenylethylthio andchlorophenylethylthio.

“Arylalkylthioalkyl” refers to an arylalkylthio group, as definedherein, to which is appended an alkyl group, as defined herein.Exemplary arylalklythioalkyl groups include benzytthiomethyl,phenylethyithiomethyl and chlorophenylethylthioethyl.

“Alkylthioalkyl” refers to an alkylthio group, as defined herein, towhich is appended an alkyl group, as defined herein. Exemplaryalkylthioalkyl groups include allylthiomethyl, ethylthiomethyl andtrifluoroethylthiomethyl.

“Alkoxyalkyl” refers to an alkoxy group, as defined herein, appended toan alkyl group, as defined herein. Exemplary alkoxyalkyl groups includemethoxymethyl, methoxyethyl and isopropoxymethyl.

“Alkoxyhaloalkyl” refers to an alkoxy group, as defined herein, appendedto a haloalkyl group, as defined herein. Exemplary alkoxyhaloalkylgroups include 4-methoxy-2-chlorobutyl.

“Cycloalkoxy” refers to R₅₄O—, wherein R₅₄ is a cycloalkyl group or abridged cycloalkyl group, as defined herein. Exemplary cycloalkoxygroups include cyclopropyloxy, cyclopentyloxy and cyclohexyloxy.

“Cycloalkylthio” refers to R₅₄S—, wherein R₅₄ is a cycloalkyl group or abridged cycloalkyl group, as defined herein. Exemplary cycloalkylthiogroups include cyclopropylthio, cyclopentytthio and cyclohexyithio.

“Haloalkoxy” refers to an alkoxy group, as defined herein, in which oneor more of the hydrogen atoms on the alkoxy group are substituted withhalogens, as defined herein. Exemplary haloalkoxy groups include1,1,1-trichloroethoxy and 2-bromobutoxy.

“Hydroxy” refers to —OH.

“Oxy” refers to —O—.

“Oxo” refers to ═O.

“Oxylate” refers to —O⁻ R₇₇ ⁺ wherein R₇₇ is an organic or inorganiccation.

“Thiol” refers to —SH.

“Thio” refers to —S—.

“Oxime” refers to ═N—OR₈₁ wherein R⁸¹ is a hydrogen, an alkyl group, anaryl group, an alkylsulfonyl group, an arylsulfonyl group, a carboxylicester, an alkylcarbonyl group, an arylcarbonyl group, a carboxamidogroup, an alkoxyalkyl group or an alkoxyaryl group.

“Hydrazone” refers to ═N—N(R₈₁)(R′₈₁) wherein R₈₁ is independentlyselected from R₆₁, and R₈₁ is as defined herein.

“Hydrazino” refers to H₂N—N(H)—.

“Organic cation” refers to a positively charged organic ion. Exemplaryorganic cations include alkyl substituted ammonium cations.

“Inorganic cation” refers to a positively charged metal ion. Exemplaryinorganic cations include Group I metal cations such as for example,sodium, potassium, magnesium and calcium.

“Hydroxyalkyl” refers to a hydroxy group, as defined herein, appended toan alkyl group, as defined herein.

“Nitrate” refers to —O—NO₂ i.e. oxidized nitrogen.

“Nitrite” refers to —O—NO i.e. oxidized nitrogen.

“Nitro” refers to the group —NO₂ and “nitrosated” refers to compoundsthat have been substituted therewith.

“Nitroso” refers to the group —NO and “nitrosylated” refers to compoundsthat have been substituted therewith.

“Nitrile” and “cyano” refer to —CN.

“Halogen” or “halo” refers to iodine (I), bromine (Br), chlorine (Cl),and/or fluorine (F).

“Imine” refers to —C(═N—R₅₁)— wherein R₅₁ is a hydrogen atom, an alkylgroup, an aryl group or an arylheterocyclic ring, as defined herein.

“Amine” refers to any organic compound that contains at least one basicnitrogen atom.

“Amino” refers to —NH₂, an alkylamino group, a dialkylamino group, anarylamino group, a diarylamino group, an alkylarylamino group or aheterocyclic ring, as defined herein.

“Alkylamino” refers to R₅₀NH—, wherein R₅₀ is an alkyl group, as definedherein. Exemplary alkylamino groups include methylamino, ethylamino,butylamino, and cyclohexylamino.

“Arylamino” refers to R₅₅NH—, wherein R₅₅ is an aryl group, as definedelsewhere herein.

“Dialkylamino” refers to R₅₂R₅₃N—, wherein R₅₂ and R₅₃ are eachindependently an alkyl group, as defined herein. Exemplary dialkylaminogroups include dimethylamino, diethylamino and methyl propargylamino.

“Diarylamino” refers to R₅₅R₆₀N—, wherein R₅₅ and R₆₀ are eachindependently an aryl group, as defined herein.

“Alkylarylamino” or “arylalkylamino” refers to R₅₂R₅₅N—, wherein R₅₂ isan alkyl group, as defined herein, and R₅₅ is an aryl group, as definedherein.

“Alkylarylalkylamino” refers to R₅₂R₇₉N—, wherein R₅₂ is an alkyl group,as defined herein, and R₇₉ is an arylalkyl group, as defined herein.

“Alkylcycloalkylamino” refers to R₅₂R₈₀N—, wherein R₅₂ is an alkylgroup, as defined herein, and R₈₀ is a cycloalkyl group, as definedherein.

“Aminoalkyl” refers to an amino group, an alkylamino group, adialkylamino group, an arylamino group, a diarylamino group, analkylarylamino group or a heterocyclic ring, as defined herein, to whichis appended an alkyl group, as defined herein. Exemplary aminoalkylgroups include dimethylaminopropyl, diphenylaminocyclopentyl andmethylaminomethyl.

“Aminoaryl” refers to an aryl group to which is appended an alkylaminogroup, an arylamino group or an arylalkylamino group. Exemplaryaminoaryl groups include anilino, N-methylanilino and N-benzylanilino.

“Thio” refers to —S—.

“Sulfinyl” refers to —S(O)—.

“Methanthial” refers to —C(S)—.

“Thial” refers to ═S.

“Sulfonyl” refers to —S(O)₂ ⁻.

“Sulfonic acid” refers to —S(O)₂OR₇₆, wherein R₇₆ is a hydrogen, anorganic cation or an inorganic cation, as defined herein.

“Alkylsulfonic acid” refers to a sulfonic acid group, as defined herein,appended to an alkyl group, as defined herein.

“Arylsulfonic acid” refers to a sulfonic acid group, as defined herein,appended to an aryl group, as defined herein.

“Sulfonic ester” refers to —S(O)₂OR₅₈, wherein R₅₈ is an alkyl group, anaryl group, or an aryl heterocyclic ring, as defined herein.

“Sulfonamido” refers to —S(O)₂—N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ when takentogether are a heterocyclic ring, a cycloalkyl group or a bridgedcycloalkyl group, as defined herein.

“Alkylsulfonamido” refers to a sulfonamido group, as defined herein,appended to an alkyl group, as defined herein.

“Arylsulfonamido” refers to a sulfonamido group, as defined herein,appended to an aryl group, as defined herein.

“Alkylthio” refers to R₅₀S—, wherein R₅₀ is an alkyl group, as definedherein (preferably a lower alkyl group, as defined herein).

“Arylthio” refers to R₅₅S—, wherein R₅₅ is an aryl group, as definedherein.

“Arylalkylthio” refers to an aryl group, as defined herein, appended toan alkylthio group, as defined herein.

“Alkylsulfinyl” refers to R₅₀—S(O)—, wherein R₅₀ is an alkyl group, asdefined herein.

“Alkylsulfonyl” refers to R₅₀—S(O)₂—, wherein R₅₀ is an alkyl group, asdefined herein.

“Alkylsulfonyloxy” refers to R₅₀—S(O)₂—O—, wherein R₅₀ is an alkylgroup, as defined herein.

“Arylsulfinyl” refers to R₅₅—S(O)—, wherein R₅₅ is an aryl group, asdefined herein.

“Arylsulfonyl” refers to R₅₅—S(O)₂—, wherein R₅₅ is an aryl group, asdefined herein.

“Arylsulfonyloxy” refers to R₅₅—S(O)₂—O—, wherein R₅₅ is an aryl group,as defined herein.

“Amidyl” refers to R₅₁C(O)N(R₅₇)— wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein.

“Ester” refers to R₅₁C(O)R₈₂— wherein R₅₁ is a hydrogen atom, an alkylgroup, an aryl group or an arylheterocyclic ring, as defined herein andR₈₂ is oxygen or sulfur.

“Carbamoyl” refers to —O—C(O)N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ taken togetherare a heterocyclic ring, a cycloalkyl group or a bridged cycloalkylgroup, as defined herein.

“Carboxyl” refers to —C(O)OR₇₆, wherein R₇₆ is a hydrogen, an organiccation or an inorganic cation, as defined herein.

“Carbonyl” refers to —C(O)—.

“Alkylcarbonyl” refers to R₅₂—C(O)—, wherein R₅₂ is an alkyl group, asdefined herein.

“Arylcarbonyl” refers to R₅₅—C(O)—, wherein R₅₅ is an aryl group, asdefined herein.

“Arylalkylcarbonyl” refers to R₅₅—R₅₂—C(O)—, wherein R₅₅ is an arylgroup, as defined herein, and R₅₂ is an alkyl group, as defined herein.

“Alkylarylcarbonyl” refers to R₅₂—R₅₅—C(O)—, wherein R₅₅ is an arylgroup, as defined herein, and R₅₂ is an alkyl group, as defined herein.

“Heterocyclicalkylcarbonyl” refer to R₇₈C(O)— wherein R₇₈ is aheterocyclicalkyl group, as defined herein.

“Carboxylic ester” refers to —C(O)OR₅₈, wherein R₅₈ is an alkyl group,an aryl group or an aryl heterocyclic ring, as defined herein.

“Alkylcarboxylic acid” and “alkylcarboxyl” refer to an alkyl group, asdefined herein, appended to a carboxyl group, as defined herein.

“Alkylcarboxylic ester” refers to an alkyl group, as defined herein,appended to a carboxylic ester group, as defined herein.

“Alkyl ester” refers to an alkyl group, as defined herein, appended toan ester group, as defined herein.

“Arylcarboxylic acid” refers to an aryl group, as defined herein,appended to a carboxyl group, as defined herein.

“Arylcarboxyliic ester” and “arylcarboxyl” refer to an aryl group, asdefined herein, appended to a carboxylic ester group, as defined herein.

“Aryl ester” refers to an aryl group, as defined herein, appended to anester group, as, defined herein.

“Carboxamido” refers to —C(O)N(R₅₁)(R₅₇), wherein R₅₁ and R₅₇ are eachindependently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ when takentogether are a heterocyclic ring, a cycloalkyl group or a bridgedcycloalkyl group, as defined herein.

“Alkylcarboxamido” refers to an alkyl group, as defined herein, appendedto a carboxamido group, as defined herein.

“Arylcarboxamido” refers to an aryl group, as defined herein, appendedto a carboxamido group, as defined herein.

“Urea” refers to —N(R₅₉)—C(O)N(R₅₁)(R₅₇) wherein R₅₁, R₅₇, and R₅₉ areeach independently a hydrogen atom, an alkyl group, an aryl group or anarylheterocyclic ring, as defined herein, or R₅₁ and R₅₇ taken togetherare a heterocyclic ring, a cycloalkyl group or a bridged cycloalkylgroup, as defined herein.

“Phosphoryl” refers to —P(R₇₀)(R₇₁)(R₇₂), wherein R₇₀ is a lone pair ofelectrons, thial or oxo, and R₇₁ and R₇₂ are each independently acovalent bond, a hydrogen, a lower alkyl, an alkoxy, an alkylamino, ahydroxy, an oxy or an aryl, as defined herein.

“Phosphoric acid” refers to —P(O)(OR₅₁)OH wherein R₅₁ is a hydrogenatom, an alkyl group, an aryl group or an arylheterocyclic ring, asdefined herein.

“Phosphinic acid” refers to —P(O)(R₅₁)OH wherein R₅₁ is a hydrogen atom,an alkyl group, an aryl group or an arylheterocyclic ring, as definedherein.

“Silyl” refers to —Si(R₇₃)(R₇₄)(R₇₅), wherein R₇₃, R₇₄ and R₇₅ are eachindependently a covalent bond, a lower alkyl, an alkoxy, an aryl or anarylalkoxy, as defined herein.

“Organic acid” refers to compound having at least one carbon atom andone or more functional groups capable of releasing a proton to a basicgroup. The organic acid preferably contains a carboxyl, a sulfonic acidor a phosphoric acid moeity. Exemplary organic acids include aceticacid, benzoic acid, citric acid, camphorsulfonic acid, methanesulfonicacid, taurocholic acid, chlordronic acid, glyphosphate and medronicacid.

“Inorganic acid” refers to a compound that does not contain at least onecarbon atom and is capable of releasing a proton to a basic group.Exemplary inorganic acids include hydrochloric acid, sulfuric acid,nitric acid and phosphoric acid.

“Organic base” refers to a carbon containing compound having one or morefunctional groups capable of accepting a proton from an acid group. Theorganic base preferably contains an amine group. Exemplary organic basesinclude triethylamine, benzyldiethylamine, dimethylethyl amine,imidazole, pyridine and pipyridine.

“Independently selected” groups are groups present in the same structurethat need not all represent the same substitution. For example, wheretwo substituents are represented as NOR_(A) and each R_(A) is said to beindependently selected from H, methyl, ethyl, etc., this means thatwhere one R_(A) is methyl, the other R_(A) may be methyl but could be Hor ethyl (or any other recited substitution).

Some of the compounds for use in the methods of the present inventionmay contain one or more chiral centers and therefore may exist inenantiomeric and diastereomeric forms. The scope of the presentinvention is intended to cover use of all isomers per se, as well asmixtures of cis and trans isomers, mixtures of diastereomers and racemicmixtures of enantiomers (optical isomers) as well. Further, it ispossible using well known techniques to separate the various forms, andsome embodiments of the invention may feature purified or enrichedspecies of a given enantiomer or diastereomer.

A “pharmacological composition” refers to a mixture of one or more ofthe compounds described herein, or pharmaceutically acceptable saltsthereof, with other chemical components, such as pharmaceuticallyacceptable carriers and/or excipients. The purpose of a pharmacologicalcomposition is to facilitate administration of a compound to anorganism.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject agent fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;phosphate buffer solutions; and other non-toxic compatible substancesemployed in pharmaceutical formulations. A physiologically acceptablecarrier should not cause significant irritation to an organism and doesnot abrogate the biological activity and properties of the administeredcompound.

A “solvate” is a complex formed by the combination of a solute (e.g., ametalloprotease inhibitor) and a solvent (e.g., water). See J. Honig etal., The Van Nostrand Chemist's Dictionary, p. 650 (1953).

The terms “optical isomer”, “geometric isomer” (e.g., a cis and/or transisomer), “stereoisomer”, and “diastereomer” have the accepted meanings(see, e.g., Hawley's Condensed Chemical Dictionary, 11th Ed.). Theillustration of specific protected forms and other derivatives of thecompounds of the instant invention is not intended to be limiting. Theapplication of other useful protecting groups, salt forms, prodrugsetc., is within the ability of the skilled artisan.

A “prodrug” is a form of a drug that must undergo chemical conversion bymetabolic processes before becoming an active, or fully active,pharmacological agent. A prodrug is not active, or is less active, inits ingested or absorbed or otherwise administered form. For example, aprodrug may be broken down by bacteria in the digestive system intoproducts, at least one of which will become active as a drug.Alternatively, it may be administered systemically, such as byintravenous injection, and subsequently be metabolized into one or moreactive molecules.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been found that certainsmall molecule ligands are capable of reversibly binding non-covalentlyto the opsin protein and inhibiting the binding of 11-cis-retinal, to anopsin retinal binding pocket. Such interference with retinal bindingreduces the formation of visual cycle products, such asall-trans-retinal, and thereby inhibits the production of compounds suchas lipofuscin and A2E with resulting reduced risk and occurrence oftoxicity that can result from accumulation of these substances. Suchcompounds, acting as pharmacologic chaperones, are also able tofacilitate the proper folding and trafficking of mutant opsinsassociated with RP. Additionally, by inhibiting 11-cis-retinal bindingand rhodopsin formation, the excessive stimulation and resultingactivation of rhodopsin caused by exposure of the retina to bright lightespecially during retinal surgery reduces photocell death.

Certain synthetic retinoids (compounds structurally related to retinol(Vitamin A alcohol)) have been reported to bind to opsin. In theembodiments of the present invention, non-retinoid small molecules(compounds having a molecular weight less than about 1000 daltons, lessthan 800, less than 600, less than 500, less than 400, or less thanabout 300 daltons) have been found to bind to opsin.

The invention features compositions and methods that are useful forreducing formation of visual cycle products and toxicity associated withthe accumulation of such products in vivo, reducing the probability ofapoptotic events associated with excessive rhodopsin activation as wellas preventing rod cell death due to aberrant processing and traffickingof mutant opsin proteins associated with RP.

Mislocalization of photoreceptor cell visual pigment proteins (opsins)can occur in various ocular diseases, and also with normal aging. Insuch cases the accumulation of mislocalized opsin leads to the declinein viability of photoreceptor cells. With time this mislocalized opsinaccumulation leads to rod and cone cell death, retinal degeneration, andloss of vision.

In one aspect, the invention provides a method of correctingmislocalized opsin within a photoreceptor cell, comprising contacting amislocalized opsin protein with an opsin-binding agent that bindsreversibly and/or non-covalently to said mislocalized opsin protein,thereby promoting correct intracellular processing and transport of saidopsin protein. Such opsin-binding agent is referred to as a “ProductiveChaperone.”

Such correction of mislocalization reduces photoreceptor cell stress,preventing photoreceptor cell decline in viability and death in variousdiseases of vision loss, and in normal age-related decline in dim-lightand peripheral rod-mediated vision, central cone-mediated vision, andloss of night vision.

In another aspect of the invention, the opsin-binding agent promotes thedegradation of the mislocalized opsin protein. This type ofopsin-binding agent is referred to as a “Counterproductive”, Shipwreck”,or “Destructive Chaperone.”

Enhancing the degradation of the mislocalized opsin by such an agentreduces the amount of mislocalized protein, thereby relievingphotoreceptor cell stress, preventing decline in viability and death ofphotoreceptor cells in diseases of vision loss, as well as in normalage-related decline in dim-light and peripheral rod-mediated vision,central cone-mediated vision, and loss of night vision.

In embodiments of the foregoing, the ophthalmic condition is one or moreof wet or dry form of macular degeneration, retinitis pigmentosa, aretinal or macular dystrophy, Stargardt's disease, Sorsby's dystrophy,autosomal dominant drusen, Best's dystrophy, peripherin mutationassociate with macular dystrophy, dominant form of Stargart's disease,North Carolina macular dystrophy, light toxicity, retinitis pigmentosa,normal vision loss related aging and normal loss of night vision relatedto aging.

Opsin, the GPCR (G-protein coupled receptor) responsible for vision,readily regenerates with 11-cis-retinal to form the visual pigmentrhodopsin.

The pigment is generated by formation of a protonated Schiff basebetween the aldehyde group of 11-cis-retinal and the ε-amino group ofL-lysine in opsin (Matsumoto and Yoshizawa, Nature 1975 Dec. 11;258(5535):523-6).

Thus, the present invention provides compositions and methods of use ofsmall molecule compounds that bind to wild type and mutant opsins andcompete with, or other wise prevent, 11-cis-retinal from combining withopsin to form rhodopsin and thereby inhibit formation of 11-cis-retinaland other visual cycle products.

Binding to this site may be predicted by the efficiency upon which theligand is able to displace and/or replace the waters in the varioushydration sites in the 11-cis retinal binding pocket as defined by thewater map technology. Hydration sites labeled with an “R” (FIG. 1 showshydration sites as circles or spheres) that are occupied by waters thatare predicted to have hydrogen bonding interactions with the protein.Thus, ligands that displace these waters will ideally have functionalitysuitably oriented when the ligand binds to replace those hydrogen bondsthat are broken in the process of the compound occupying the bindingpocket.

In accordance with the present invention, ligand binding potency isenhanced by compounds that efficiently displace highly unstable watersfrom the opsin binding pocket. Occupation of the pocket by apharmacologic chaperone creates interactions between the ligand and theprotein which induce the proper folding and/or stabilization of thenative 3-dimensional conformation of the protein that leads to it beingproperly processed and trafficked to its proper location in the cellmembrane.

Alternatively, hydration sites labeled with a “D” (FIG. 1) locate watersthat are in hydrophobic environments and therefore it is optimal for thebinding compound to displace all of these waters with nonpolarsubstituents that compliment the hydrophobic environment of the protein.Thus, displacing waters in hydrophobic enviromments while replacing thehydrogen bonds of waters in hydration sites redicted to have hydrogenbonding interactions with the protein with functionality on the ligandthat can act as water mimetics when these waters are displaced leads tooptimal potency and efficacy. Alternatively, displacing waters inhydration sites labeled with a “D” in FIG. 1 and leaving those waters inhydration sites labeled with an “R: (shown in FIG. 1) unperturbed suchthat their environment with the ligand bound does not adversely affectthe intrinsic stability of these waters in the pocket in the absence ofligand occupation leads to potent and efficacious compounds. Thehydration sites are predicted locations of waters in the absence of aligand based on the hydration map. Binding of a ligand of the inventionmay follow one of four possible mechanisms: (i) displacing a wateroccupying a hydration site, (ii) replacing a hydrogen bond betweenprotein and a water in a hydration site by a functionality of theligand, (iii) binding of a ligand and leaving a water in the hydrationsite intact, and (iv) forming an extended hydrogen bonding network withthe water in a hydration site while not displacing it.

In one embodiment, the invention provides opsin binding ligands ofFormula (I) and pharmaceutically acceptable salts thereof:

A-B-Q-V   Formula I

wherein A is:

B is

-   -   1) —(CH₂)_(n)—;    -   2) —CH═CH—;    -   3) —CH₂N(R²²)—;    -   4) —CH₂—O—    -   5) —C(O)—CH₂—C(O)—    -   6)

or

-   -   7) —C(O)NR²²—;

wherein n=0, 1 or 2 and

E is:

-   -   1) —N(R²²)—; or    -   2) oxygen;

Q is:

-   -   1) —C(O)—;    -   2) —(CH₂)_(a)—;    -   3) —S(O₂)—; or    -   4) —CH₂—C(O)—

wherein a is 1 or 2;

V is:

wherein b is 1 or 2 and a is 1 or 2;

Y is:

-   -   1) NR²²;    -   2) N-Q-U;    -   3) CR²²R²³;    -   4) oxygen;    -   5) S(O)_(n);    -   6) N—C(S)—NR²²R²³;    -   7) N—(C═N—CN)—NR²²R²³;    -   8) N—(C═N—SO₂CH₃)—NR²²R²³;    -   9) C═NOR²²;    -   10) C═N—NR²²R²³; or    -   11) C-Q-U;

and n is 0, 1 or 2;

U is:

-   -   1) NR²²R²³;    -   2) lower alkyl;    -   3) haloalkyl;    -   4) alkoxy;    -   5) OR²²; or    -   6) hydrogen;

X is:

-   -   1) hydrogen    -   2) alkyl; or    -   3) —C═CR⁹;

R¹ and R² are independently:

-   -   1) —CH₃; or    -   2) —CH₂CH₃;

R³ is:

-   -   1) hydrogen;    -   2) —CH₃; or    -   3) —CH₂CH₃;

R_(a), and R_(b), are each independently:

-   -   1) hydrogen;    -   2) deutero; or    -   3) —CH₃

R_(c), and R_(d), are each independently:

-   -   1) hydrogen;    -   2) alkoxy;    -   3) lower alkyl; or    -   4) alkenyl;

R⁴ is:

-   -   1) —CH₃;    -   2) —CF₃;    -   3) —C₂H₅; or    -   4) —C₃H₅;

R⁵, R⁶ and R⁷ are each independently:

-   -   1) hydrogen;    -   2) lower alkyl;    -   3) halogen;    -   4) dialkylamine;    -   5) nitro; or    -   6) dialkylamine;

Z is:

-   -   1) CR³;    -   2) CH; or    -   3) nitrogen;

R⁸ is:

-   -   1) —CH₂—; or    -   2) —C(O)—;

R⁹, R¹⁴ and R¹⁶ are each independently:

-   -   1) hydrogen; or    -   2) —CH₃;

R¹⁰ is:

-   -   1) N—R¹³;    -   2) sulfur; or    -   3) oxygen;

R¹¹ is:

-   -   1) ═N—; or    -   2) ═C(CH₃)—;

R¹² is:

-   -   1) lower alkyl;    -   2) alkoxy; or    -   3) haloalkyl;

R¹³ is:

-   -   1) phenyl;    -   2) lower alkyl; or    -   3) haloalkyl;

R¹⁵ is:

-   -   1) hydrogen; or    -   2) —C(O)CH₃;

R¹⁷ and R¹⁸ together are:

-   -   1) —(CH₂)₄—; or    -   2) —CH═CH—CH═CH—

R¹⁹ and R²⁰ together are:

-   -   1) —CH₂—C(CH₃)₂—CH₂—C(O)—; or    -   2) —CH═CH—CH═CH—;

R²¹ is:

-   -   1) hydrogen;    -   2) —C(O)CH₃;    -   3) —CH₃; or    -   4) —CH₂CH₃;

R²² and R²³ are each independently:

-   -   1) hydrogen; or    -   2 lower alkyl;

R²⁴ and R^(2s) are each independently:

-   -   1) hydrogen; or    -   2) —CH₃;

R²⁶ is:

-   -   1) NR²²R²³: or    -   2) alkoxy;

And wherein R¹ and R² taken together or R_(a) and R_(b) taken togetheralong with the carbon to which they are attached can form cyclopropyl;

R²⁴ and R²⁵ taken together along with the two carbons to which they areattached can form cyclopropyl:

R²⁴ and R²⁵ taken together can form oxo;

And wherein T is:

-   -   1) oxygen;    -   2) —N(R¹⁶)—; or    -   3) sulfur;

E is:

-   -   1) oxygen;    -   2) —N(R¹⁶)—;    -   3) sulfur; or    -   4) —C(O)—.

In its broadest embodiments, R¹, R² and R³ are each independently loweralkyl.

In preferred embodiments, the compound has the structure of Formula Iwherein V is:

and wherein a and b are each independently 1 or 2, more preferablywherein at least one of a or b is 1, most preferably wherein both a andb are 1, X is hydrogen, R²³, R²⁴ and R²⁵ are hydrogen, Y isC—C(O)NR²²R²³ or N—C(O)NR²²R²³ and R²² and R²³ are both hydrogen.

In preferred examples of the invention, the compound has the structureof Formula I wherein A is:

In preferred embodiments thereof, one or more of R¹ and R² is methyl,more preferably both are methyl, and R³ is a hydrogen or a methyl group.In other specific embodiments, R_(a) and R_(b) are independentlyhydrogen, deutero or methyl, preferably hydrogen or methyl, R_(c) andR_(d) are preferably hydrogen or lower alkyl, most preferably hydrogenor methyl.

In another preferred example, the compound has the structure of FormulaI wherein A is:

In preferred embodiments thereof, R¹, R² and R³ are each methyl, and R²⁴is a methyl or hydrogen, preferably a hydrogen.

In other specific embodiments, R_(a) and R_(b) are independentlyhydrogen, deutero or methyl, preferably hydrogen or methyl, R_(c) andR_(d) are hydrogen lower alkyl, alkoxy or alkoxymethyl, more preferablyhydrogen, alkoxy or lower alkyl, most preferably hydrogen.

In another preferred example, the compound has Formula I wherein B is—CH═CH—, —CH₂—CH₂— or —CH₂—N(R²²)—, preferably —CH═CH or —CH₂—CH₂—, andmost preferably —CH═CH—.

In another preferred example, the compound has Formula I wherein Q is—C(O)— or —CH₂—, most preferably —C(O)—.

In another preferred example, the compound has Formula I wherein X ishydrogen, lower alkyl or —C≡CR⁹, more preferably hydrogen or —C≡CR⁹wherein R⁹ is hydrogen or methyl.

In another preferred example, the compound has Formula I wherein Y isoxygen or N—C(O)—NR²²R²³, more preferably N—C(O)—NR²²R²³, mostpreferably N—C(O)—NR²²R²³ wherein R²² and R²³ are hydrogen.

In another preferred example, the compound has Formula I wherein R²⁴ andR²⁵ are each hydrogen.

In another embodiment, the invention provides opsin binding ligands ofFormula (II) and pharmaceutically acceptable salts thereof:

wherein W is:

-   -   1) —OR²²;    -   2) —NR²²R²³;    -   3) —N(R²²)—C(O)—NR²²R²³;    -   4) —O—C(O)—NR²²R²³;    -   5) —N(R²²)—C(S)—NR²²R²³;    -   6) —O—C(S)—NR²²R²³;    -   7) —S—C(O)—NR²²R²³;    -   8) —N(R²²)—(C═N—CN)—NR²²R²³;    -   9) —N(R²²)—(C═N—SO₂Me)-NR²²R²³; or    -   10) —C(O)N(R⁹)N(R¹⁴)(R¹⁶);

In preferred examples, the compound has the structure of Formula IIwherein A is:

In preferred embodiments thereof, one or more of R¹ and R² is a methylor ethyl group, preferably a methyl group, and R³ is a hydrogen or amethyl group. In other specific embodiments, R_(a) and R_(b) areindependently hydrogen, deutero or methyl, preferably hydrogen ormethyl, R_(c) and R_(d) are hydrogen lower alkyl, alkoxy oralkoxymethyl, more preferably hydrogen, alkoxy or lower alkyl, mostpreferably hydrogen.

In another preferred example, the compound has Formula II wherein A is:

In preferred embodiments thereof, R¹, R², and R³ is a ethyl or methylgroup, more preferably a methyl group and R²⁴ is a methyl or hydrogen,preferably a hydrogen. In other specific embodiments, R_(a) and R_(b)are independently hydrogen, deutero or methyl, preferably hydrogen ormethyl, R_(c) and R_(d) are hydrogen lower alkyl, alkoxy oralkoxymethyl, more preferably hydrogen, alkoxy or lower alkyl, mostpreferably hydrogen.

In another preferred example, the compound has Formula II wherein B is—CH═CH—, —CH₂—CH₂— or —CH₂—N(R²²)—, preferably —CH═CH or —CH₂—CH₂—, andmost preferably —CH═CH—.

In another preferred example, the compound has Formula II wherein Q is—C(O)— or —CH₂— most preferably —C(O)—.

In another preferred example, the compound has Formula II wherein p is 0or 1, most preferably 1.

In another preferred example, the compound has Formula II wherein W is—O—C(O)—NR²²R²³ or —N(R⁹)—C(O)—NR²²R²³, more preferably—N(R⁹)—C(O)—NR²²R²³, most preferably —N(R⁹)—C(O)—NR²²R²³ wherein each ofR⁹, R²² and R²³ is hydrogen.

In specific embodiments the opsin binding compound of Formula I orFormula (II) is (wherein each compound number corresponds to the numberof the example where it was prepared):

-   (E)-3-(2,6,6-Trimethylcyclohex-1-enyl)acrylamide (Compound 2b);-   (E)-N-Methyl-3-(2,6,6-trmethylcyclohex-1-enyl)acrylamide (Compound    3);-   (E)-N,N-Dimethyl-3-(2,6,6-trimethylcyclohex-1-enyl)acrylamide    (Compound 4);-   (E)-1-(Piperidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 5);-   (E)-1-Morpholino-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 6);-   (E)-tert-Butyl    4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1    carboxylate (Compound 7a);-   (E)-1-(1,4-Diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 7b);-   (E)-1-(4-Methyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 7c);-   (E)-1-(4-Ethyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)    prop-2-en-1-one (Compound 8);-   (E)-1-(4-propyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)    prop-2-en-1-one (Compound 9);-   (E)-1-(4-Acetyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)    prop-2-en-1-one (Compound 10);-   (E)-1-(4-Propionyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1    enyl)prop-2-en-1-one (Compound 11);-   (E)-1-(4-(2,2,2-Trifluoroacetyl)-1,4-diazepan-1-yl)-3-(2,6,6-trimethyl    cyclohex-1-enyl)prop-2-en-1-one (Compound 12);-   (E)-4-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide    (Compound 13);-   (E)-N-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide    (Compound 14);-   (E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide    (Compound 15);-   (E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide    (Compound 16);-   (E)-N-Isopropyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide    (Compound 17);-   (E)-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxylate    (Compound 18);-   (E)-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxylate    (Compound 19);-   (E)-1-(4-(Methylsulfonyl)-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclo-hex-1-enyl)prop-2-en-1-one    (Compound 20);-   (E)-1-(4-(Ethylsulfonyl)-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 21);-   (E)-1-(4-(Trifluoromethylsulfonyl)-1,4-diazepan-1-yl)-3-(2,6,6-tri-methylcyclohex-1-enyl)prop-2-en-1-one    (Compound 22);-   (E)-N-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothioamide    (Compound 23);-   (E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothioamide    (Compound 24);-   (E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothioamide    (Compound 25);-   (E)-N-Isopropyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothioamide    (Compound 26);-   (E)-1-(4-Methylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 27),-   (E)-tert-Butyl 4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)    piperazine-1-carboxylate (Compound 28a);-   (E)-1-(Piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 28b);-   (E)-1-(4-Ethylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 28c);-   (E)-1-(4-Propylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 29),-   (E)-1-(4-Acetylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 30);-   (E)-1-(4-Propionylpiperazin-1-yl)-3-(2,6,6-trmethylcyclohex-1-enyl)    prop-2-en-1-one (Compound 31);-   (E)-1-(4-(2,2,2-Trifluoroacetyl)    piperazin-1-yl)-3-(2,6,6-trimethylcyclo-hex-1-enyl)prop-2-en-1-one    (Compound 32);-   (E)-4-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 33);-   (E)-N-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 34);-   (E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 35);-   (E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 36);-   (E)-N-Isopropyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 37);-   (E)-Methyl 4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)    piperazine-1-carboxylate (Compound 38);-   (E)-Ethyl 4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)    piperazine-1-carboxylate (Compound 39);-   (E)-1-(4-(Methylsulfonyl)piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 40);-   (E)-1-(4-(Ethylsulfonyl)piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 41);-   (E)-1-(4-(Trifluoromethylsulfonyl)piperazin-1-yl)-3-(2,6,6-trimethyl-cyclohex-1-enyl)prop-2-en-1-one    (Compound 42);-   (E)-N-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide    (Compound 43);-   (E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide    (Compound 44);-   (E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide    (Compound 45);-   (E)-N-Isopropyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide    (Compound 46);-   (S,E)-1-(3-Hydroxypyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)    prop-2-en-1-one (Compound 47);-   (S,E)-1-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)pyrrolidin-3-yl    carbamate (Compound 48);-   (E)-tert-Butyl 1-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)    pyrrolidin-3-yl carbamate (Compound 49a);-   (E)-1-(3-Aminopyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 49b);-   (E)-1-(1-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)    pyrrolidin-3-yl) urea (Compound 50);-   1-(Piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)propan-1-one    (Compound 51a);-   4-(3-(2,6,6-Trimethylcyclohex-1-enyl)propanoyl)piperazine-1-carboxamide    (Compound 51b);-   (S,E)-2-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 52);-   (R,E)-2-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 53);-   (E)-N²-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1,2-dicarboxamide    (Compound 54);-   N¹-((2,6,6-Trimethylcyclohex-1-en-1-yl)methyl)piperazine-1,4-dicarboxamide    (Compound 55);-   N¹-Methyl-N¹-((2,6,6-trimethylcyclohex-1-en-1-yl)methyl)piperazine-1,4-dicarboxamide    (Compound 56);-   (R,E)-1-(3-Hydroxypyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one    (Compound 57a);-   (R,E)-1-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)    pyrrolidin-3-yl carbamate (Compound 57b);-   (S,E)-1-(3-Aminopyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one    (Compound 58b);-   (S,E)-1-(1-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)pyrrolidin-3-yl)urea    (Compound 58c);-   (R,E)-1-(3-Aminopyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one    (Compound 59b);-   (R,E)-1-(1-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)    pyrrolidin-3-yl)urea (Compound 59c);-   (E)-4-(3-(2,6,6-Trimethyl-3-oxocyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide    (Compound 60);-   (E)-4-(3-(3,3-Difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide    (Compound 62);-   (E)-4-(3-(3,3-Dideutero-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)    piperazine-1-carboxamide (Compound 63);-   (E)-1-(1,1-Dioxidothiomorpholino)-3-(2,6,6-trimethylcyclohex-1-eyl)prop-2-en-1-one    (Compound 68);-   (E)-1-Thiomorpholino-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one    (Compound 69);-   (E)-1-(4,4-Difluoropiperidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one    (Compound 70);-   (+)-4-((E)-3-((1,6-anti)-2,2,6-trimethylcyclohexyl)    acryloyl)piperazine-1-carboxamide (Compound 71);-   (−)-4-((E)-3-((1R,6R)-2,2,6-trimethylcyclohexyl)acryloyl)    piperazine-1-carboxamide (Compound 72);-   (+)-4-((E)-3-((1 S,6S)-2,2,6-trimethylcyclohexyl)acryloyl)    piperazine-1-carboxamide (Compound 73);-   (E)-1-Morpholino-3-((1R,6R)-2,2,6-trimethylcyclohexyl)prop-2-en-1-one    (Compound 74);-   (E)-1-Thiomorpholino-3-((1R,6R)-2,2,6-trimethylcyclohexyl)    prop-2-en-1-one (Compound 75);-   (E)-4-(3-(2,5,6,6-tetramethylcyclohex-2-en-1-yl)acryloyl)    piperazine-1-carboxamide (Compound 76);-   4-((E)-3-((1R,6S)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide    and    4-((E)-3-((1S,6R)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide    (Compound 77);-   (E)-4-(3-(2,6,6-trimethylcyclohex-2-en-1-yl)acryloyl)piperazine-1-carboxamide    (Compound 78);-   4-(3-((1R,6S)-2,2,6-trimethylcyclohexyl)propanoyl)piperazine-1-carboxamide    (Compound 80);-   4-(3-((1    S,6R)-2,2,6-trimethylcyclohexyl)propanoyl)piperazine-1-carboxamide    (Compound 81):-   (E)-1-Morpholino-3-((1    S,6S)-2,2,6-trimethylcyclohexyl)prop-2-en-1-one (Compound 83);-   (E)-4-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)piperazin-2-one    (Compound 84);-   (E)-4-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carbaldehyde    (Compound 88);-   (E)-1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one    (Compound 89);-   (±)-3,5-cis-Dimethyl-4-((E)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)    piperazine-1-carboxamide (Compound 90);-   (E)-4-(3-(2,2,6-trimethylbicyclo[4.1.0]heptan-1-yl)acryloyl)    piperazine-1-carboxamide (Compound 91);-   (±)-(E)-4-(3-(4-Methoxy-2,6,6-trimethylcyclohex-1-en-1-yl) acryloyl)    piperazine-1-carboxamide (Compound 93);-   (−)-((1R,6S)-2,2,6-trimethylcyclohexyl)methyl    4-carbamoylpiperazine-1-carboxylate (Compound 94);-   (−)-N¹-Methyl-N¹-(((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)    piperazine-1,4-dicarboxamide (Compound 95);-   N¹-(((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)piperazine-1,4-dicarboxamide    (Compound 96);-   4-(((1R,6S)-2,2,6-trimethylcyclohexanecarboxamido)methyl)piperidine-1-carboxamide    (Compound 97);-   (E)-2-(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)azetidin-3-yl)    acetamide (Compound 98);-   (E)-3-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acrylamido)    azetidine-1-carboxamide (Compound 99);-   (E)-3-(2,6,6-Trimethylcyclohex-1-en-1-yl)-N-(2-ureidoethyl)acrylamide    (Compound 100);-   (E)-N-Methyl-N-(2-(1-methylureido)ethyl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)    acrylamide (Compound 101);-   (E)-4-(3-(2,2,6,6-Tetramethylcyclohexyl)acryloyl)piperazine-1-carboxamide    (Compound 102);-   (E)-1-Morpholino-3-(2,2,6,6-tetramethylcyclohexyl)prop-2-en-1-one    (Compound 103);-   N-((2,6,6-Trimethylcyclohex-1-en-1-yl)methyl)morpholine-4-carboxamide    (Compound 104);-   (E)-4-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)    piperazine-1-carbothioamide (Compound 105);-   (E)-2-Ethynyl-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide    (Compound 106);-   (E)-1-Morpholino-3-(3,3,6,6-tetramethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 107);-   (E)-4-(3-(3,3,6,6-Tetramethylcyclohex-1-enyl)acryloyl)    piperazine-1-carboxamide (Compound 108);-   (E)-4-(3-(3,6,6-Trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide    (Compound 109); and-   (E)-1-Morpholino-3-(3,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one    (Compound 110);    including all pharmaceutically acceptable salts, hydrates, or    solvates thereof.

All Compound Names were Derived Using ChemBioDraw 11.0.1.

Especially preferred examples of the compounds of the invention, andmethods using said compounds, include compounds of Table 1, and are alsoselected from one or more of the group consisting of compounds 6, 13,14, 22, 33, 34, 37, 44, 45, 50, 51a, 51b, 52, 53, 55, 57, 60b, 63, 69,71, 72, 73, 80, 84, 105, 106, 107, 108, 109 and 110 including allpharmaceutically acceptable salts, solvates and hydrates thereof.

Another embodiment of the invention provides the opsin binding ligandmetabolites of the opsin binding compounds. These metabolites, includebut are not limited to, degradation products, hydrolysis products,gluconoride adducts and the like, of the opsin binding compounds andpharmaceutically acceptable salts thereof, of the opsin compounds.

Another embodiment of the invention provides processes for making thenovel compounds of the invention and to the intermediates useful in suchprocesses. The reactions are performed in solvents appropriate to thereagents and materials used are suitable for the transformations beingeffected. It is understood by one skilled in the art of organicsynthesis that the functionality present in the molecule must beconsistent with the chemical transformation proposed. This will, onoccasion, necessitate judgment by the routineer as to the order ofsynthetic steps, protecting groups required, and deprotectionconditions. Substituents on the starting materials may be incompatiblewith some of the reaction conditions required in some of the methodsdescribed, but alternative methods and substituents compatible with thereaction conditions will be readily apparent to one skilled in the art.The use of sulfur, nitrogen and oxygen protecting groups is well knownfor protecting thiol, amino and alcohol groups against undesirablereactions during a synthetic procedure and many such protecting groupsare known and described by, for example, Greene and Wuts, ProtectiveGroups in Organic Synthesis, Third Edition, John Wiley & Sons, New York(1999).

Compounds of the invention that have one or more asymmetric carbon atomsmay exist as the optically pure enantiomers, pure diastereomers,mixtures of enantiomers, mixtures of diastereomers, racemic mixtures ofenantiomers, diasteromeric racemates or mixtures of diastereomericracemates. It is to be understood that the invention anticipates andincludes within its scope all such isomers and mixtures thereof.

The chemical reactions described herein are generally disclosed in termsof their broadest application to the preparation of the compounds ofthis invention. Occasionally, the reactions may not be applicable asdescribed to each compound included within the disclosed scope. Thecompounds for which this occurs will be readily recognized by oneskilled in the art. In all such cases, either the reactions can besuccessfully performed by conventional modifications known to oneskilled in the art, e.g., by appropriate protection of interferinggroups, by changing to alternative conventional reagents, by routinemodification of reaction conditions, or other reactions disclosed hereinor otherwise conventional, will be applicable to the preparation of thecorresponding compounds of this invention. In all preparative methods,all starting materials are known or readily prepared from known startingmaterials.

Methods of the Invention

The present invention provides a method of using compounds of theFormula I and/or Formula II for reducing the formation of toxic visualcycle products, comprising contacting an opsin protein with smallmolecule ligands that reversibly bind to said opsin protein to inhibit1-cis-retinal binding in said binding pocket, thereby reducing formationof toxic visual cycle products associated with wet or dry ARMD. andreducing photocell apoptosis associated with excessive rhodopsinactivation as a result of bright light stimulation.

The present invention also provides a method of use of compounds of theFormula I and/or Formula II for treating, preventing or reducing therisk of light toxicity in a mammal, comprising administering to amammal, at risk of developing an ophthalmic condition that is related tothe formation or accumulation of a visual cycle product or apoptoticphotocell death.

The present invention also provides a method of use of compounds of theFormula I and/or Formula II for treating, preventing or reducing therisk of light toxicity in a mammal, comprising administering to amammal, at risk of developing an ophthalmic condition that is related tothe formation or accumulation of a visual cycle product or apoptoticphotocell death, an effective amount of a that small molecule ligandthat reversibly binds (for example, at or near the retinal bindingpocket) to an opsin protein present in the eye of said mammal, forexample, to inhibit 11-cis-retinal binding in said binding pocket,thereby reducing light toxicity and photocell apoptosis.

The present invention also provides a method of use of compounds of theFormula I and/or Formula II for treating, preventing or reducing therisk of RP in a mammal, comprising administering to a mammal, at risk ofRP related to the improper folding and trafficking of mutant opsins, aneffective amount of a that small molecule ligand that reversibly binds(for example, at or near the retinal binding pocket) to an opsin proteinpresent in the eye of said mammal, for example, to inhibit11-cis-retinal binding in said binding pocket, thereby reducing thevision loss caused by RP.

In specific examples of such methods, the small molecule ligand isselective for binding to opsin and/or the small molecule ligand binds tosaid opsin in the retinal binding pocket of said opsin protein and/orthe small molecule ligand binds to said opsin protein so as to inhibitcovalent binding of 11-cis-retinal to said opsin protein when said11-cis-retinal is contacted with said opsin protein when said smallmolecule ligand is present and/or the mammal is a human being.

In one embodiment, light toxicity is related to an ophthalmic procedure,for example, ophthalmic surgery. Said agent may be administered priorto, during or after said surgery (or at any one or more of those times).

In specific embodiments of the methods of the invention, the nativeopsin protein is present in a cell, such as a rod cell, preferably, amammalian and more preferably a human cell. In specific embodiments, thesmall molecule ligands of the invention inhibit binding of11-cis-retinal in the binding pocket of opsin and slow the visual cyclethereby reducing the formation of all-trans-retinal, or a toxic visualcycle product formed from it, such as lipofuscin orN-retinylidene-N-retinylethanolamine (A2E). Alternatively, photocellapoptosis as a result of excessive rhodopsin activation is reduced orprevented by inhibition of rhodopsin formation. Additionally, improperfolding and trafficking of mutant opsin proteins associated with RP isreduced.

In methods of the invention, administering is preferably by topicaladministration (such as with an eye wash) or by systemic administration(including oral, intraocular injection or periocular injection). By wayof preferred example, the ophthalmic condition to be treated is lighttoxicity, such as that resulting from ocular surgery, for example,retinal or cataract surgery.

Also encompassed is an ophthalmologic composition comprising aneffective amount of compounds of the Formula I and/or Formula II in apharmaceutically acceptable carrier, wherein said agent reversibly bindsnon-covalently (for example, at or near the retinal binding pocket) tosaid opsin protein to inhibit 11-cis-retinal binding in said pocket,preferably where the small molecule ligand is selective for opsinprotein.

The present invention further provides a screening method foridentifying a small molecule ligand that reduces light toxicity in amammalian eye, comprising:

(a) contacting a native opsin-protein with a test compound in thepresence of 11-cis-retinal and under conditions that promote the bindingof the test compound and the 11-cis-retinal to the native opsin protein;and

(b) determining a reversible reduction in rate of formation of rhodopsinrelative to the rate when said test compound is not present,

thereby identifying said test compound as a small molecule ligand thatreduces light toxicity in a mammalian eye. In a preferred embodiment,said test compound is structurally related to a compound disclosedherein.

In a typical competition assay of the invention, a compound is soughtthat will tie up the retinal binding pocket of the opsin protein. Thus,the assay seeks to identify a small molecule opsin binding compound (onethat will not be tightly regulated by the retina as to amount enteringrod cells) that competes with or prevents 11-cis-retinal or9-cis-retinal from forming rhodopsin or isorhodopsin. Over time, thiswill slow the rate of formation of rhodopsin relative to the rate when11-cis-retinal alone is present. In one embodiment, the assay isconducted in the presence of 11-cis-retinal, and the rate of formationof rhodopsin is measured as a way of determining competition for theretinal binding pocket, for example, by determining the rate of increasein the 500 nm peak characteristic for rhodopsin. No antibodies forrhodopsin are required for this assay. A useful compound will exhibit arate of rhodopsin formation that is at least about 2 to 5 fold lowerthan that observed in the presence of 11-cis-retinal when said testcompound is not present.

The compounds of the Formula I and/or Formula II may be administeredalong with other agents, including a mineral supplement, ananti-inflammatory agent, such as a steroid, for example, acorticosteroid, and/or an anti-oxidant. Among the corticosteroids usefulfor such administration are those selected from the group consisting ofcortisone, hydrocortisone, prednisone, prednisolone, methylprednisolone,triamcinolone, betamethasone, beclamethasone and dexamethasone. Usefulanti-oxidants include vitamin A, vitamin C and vitamin E.

The methods of the invention also contemplate reducing light toxicity byusing at least one additional agent (in addition to the compounds of theFormula I and/or Formula II selected from the group consisting of aproteasomal inhibitor, an autophagy inhibitor, a lysosomal inhibitor, aninhibitor of protein transport from the ER to the Golgi, an Hsp90chaperone inhibitor, a heat shock response activator, a glycosidaseinhibitor, and a histone deacetylase inhibitor, wherein the smallmolecule opsin binding and the additional compound are administeredsimultaneously or within fourteen days of each other in amountssufficient to treat the subject.

In a particular example of the methods of the invention, the compoundsof the Formula I and/or Formula II and the additional compound areadministered within ten days of each other, within five days of eachother, within twenty-four hours of each other and preferably areadministered simultaneously. In one example, the small molecule opsinbinding and the additional compound are administered directly to theeye. Such administration may be intraocular or intravitrial. In otherexamples, the small molecule opsin binding and the additional compoundare each incorporated into a composition that provides for theirlong-term release, such as where the composition is part of amicrosphere, nanosphere, nano emulsion or implant.

As described herein, the compounds of the Formula I and/or Formula IIuseful in the methods of the invention are available for use alone or incombination with one or more additional compounds to treat or preventconditions associated with excessive rhodopsin activation, such as lighttoxicity, for example, resulting from ocular surgical procedures. In oneembodiment, compounds of the Formula I and/or Formula II of theinvention is administered without an additional active compound. Inanother embodiment, compounds of the Formula I and/or Formula II of theinvention is used in combination and with another active compound (e.g.,as discussed herein). In still another exemplary embodiment, compoundsof the Formula I and/or Formula II are administered in combination withthe proteasomal inhibitor MG132, the autophagy inhibitor3-methyladenine, a lysosomal inhibitor ammonium chloride, the ER-Golgitransport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and the histone deacetylase inhibitor Scriptaid, can be usedto reduce formation of visual cycle products and cell apoptosis as aresult of excessive rhodopsin activation.

As described herein, the compounds of the Formula I and/or Formula IIuseful in the methods of the invention are available for use alone or incombination with one or more additional compounds to treat or preventthe aberrant processing and trafficking of mutant opsin proteinsassociated with rod cell death as a result of RP. In one embodiment,compounds of the Formula I and/or Formula II of the invention isadministered without an additional active compound. In anotherembodiment, compounds of the Formula I and/or Formula II of theinvention is used in combination and with another active compound (e.g.,as discussed herein). In still another exemplary embodiment, compoundsof the Formula I and/or Formula II are administered in combination withthe proteasomal inhibitor MG132, the autophagy inhibitor3-methyladenine, a lysosomal inhibitor ammonium chloride, the ER-Golgitransport inhibitor brefeildin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and the histone deacetylase inhibitor Scriptaid, can be usedto reduce or prevent the rod cell death and resulting blindnessassociated with RP.

As described herein, the compounds of the Formula I and/or Formula IIuseful in the methods of the invention are available for use alone or incombination with one or more additional compounds to treat or preventconditions associated with production and accumulation of toxic visualcycle products derived from all-trans-retinal, such as lipofucin andA2E, for example, the blindness associated with wet or dry ARMD. In oneembodiment, compounds of the Formula I and/or Formula II of theinvention is administered without an additional active compound. Inanother embodiment, compounds of the Formula I and/or Formula II of theinvention is used in combination and with another active compound (e.g.,as discussed herein). In still another exemplary embodiment, compoundsof the Formula I and/or Formula II are administered in combination withthe proteasomal inhibitor MG132, the autophagy inhibitor3-methyladenine, a lysosomal inhibitor ammonium chloride, the ER-Golgitransport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and the histone deacetylase inhibitor Scriptaid, can be usedto reduce formation of toxic visual cycle product metabolites and photocell death as a result of dry ARMD.

In specific embodiments of the methods of the invention, the mis-foldedopsin protein comprises a mutation in its amino acid sequence, forexample, one of the mutations T17M, P347S or P23H, preferably P23H.

Preferably, in any of the methods of the invention, the opsin-bindingagent binds to opsin in its retinal binding pocket.

In one aspect, the present invention provides a method of inhibiting theformation or accumulation of a visual cycle product, comprisingcontacting an opsin protein with a compound that reduces hydration ofsaid opsin protein, preferably wherein said compound competes with oneor more water molecules for binding to opsin. In specific embodiments ofsuch methods, the compound binds chemically to the opsin protein, forexample, through hydrogen bonding.

In specific examples of the methods of the invention, a compound usefultherein may bind to opsin at any hydration site found within the retinalbinding pocket of the opsin molecule so long as said binding excludeswholly, or in part, the binding of one or more water molecules in saidbinding pocket. Preferably the compound used in such method binds so asto occupy the left side of the binding pocket as shown in FIG. 1 anddisplace waters in hydration sites 5-20 (numbered circles in FIG. 1),more preferably binds so that waters in hydration sites 5-20 aredisplaced, and waters at hydration sites 3 or 4 as shown in FIG. 1 aredisplaced and replaced with functionality on the ligand that mimics thehydrogen bonding interactions that these waters are predicted to havewith residues on the protein.

A specific example of these methods contemplates binding of a compoundby chemical interaction with Cys187 or Glu113 of the opsin protein. Inseparate embodiments thereof, said interaction is with Cys187 or saidinteraction is with Glu113 or is with both sites. A preferred mode ofsaid interaction is hydrogen bonding.

In other specific examples, said interaction is with a carbonyl group onthe opsin protein. In specific embodiments thereof, said carbonyl is onCys187 or Glu113 of said opsin protein. Separate embodiments includewhere the carbonyl is on Cys187 of the opsin protein or where thecarbonyl is on Glu113 of the opsin protein. In one embodiment, thecarbonyl is in the gamma-carboxyl group of Glu113 of the opsin protein.A preferred embodiment is where the interaction is through an amine,carboxamido or urea group on the compound.

While use of any of the compounds disclosed herein as a means ofreducing hydration in the opsin binding pocket should be considered apreferred embodiment of such method, the reduction of formation of avisual cycle product by reducing the formation of rhodopsin is a generalmethod of the invention for reducing such visual cycle productformation, especially production of lipofuscin and/or A2E, and fortreating an ophthalmic disease by reducing said hydration is a generalaim of the invention and is not necessarily limited in scope only to theuse of chemicals disclosed herein but may include use of other known oryet to be known chemical compounds so long as they function in themethods of the invention and reduce hydration (i.e., binding of water)in the retinal binding pocket of opsin.

It should be noted that the compounds disclosed herein for use in themethods of the invention may not function to reduce hydration in theretinal binding pocket of opsin but may still function in one or more ofthe methods of the invention. For example, a compound of Formula Iand/or Formula II may bind to an allosteric site on the protein therebyexcluding retinal from the retinal binding site without necessarilydecreasing hydration yet still reduce formation of a visual cycleproduct, such as lipofuscin and/or A2E, by virtue of its excludingretinal from the binding pocket, thus non-covalently reducing theactivity of the visual cycle.

In embodiments of any of the compositions and methods of the invention,the opsin-binding agent (e.g., a non-retinoid binding agent) isselective for binding to opsin. Such selectivity is not to be taken asrequiring exclusivity that said agent may bind to other proteins as wellas to opsin but its binding to opsin will be at least selective, wherebythe binding constant (or dissociation constanti) for binding to opsinwill be lower than the average value for binding to other proteins thatalso bind retinods, such as retinal analogs. Preferably, opsin bindingagents are non-retinoid opsin-binding agents that bind non-covalently toopsin. Preferably, the opsin binding agent binds at or near the opsinretinal binding pocket, where the native ligand, 11-cis-retinal,normally binds. Without wishing to be bound by theory, in one embodimentthe binding pocket accommodates retinal or an agent of the invention,but not both. Accordingly, when an agent of the invention is bound at ornear the retinal binding pocket, other retinoids, such as11-cis-retinal, are unable to bind to opsin. Binding of an agent of theinvention inside the retinal binding pocket of a mis-folded opsinmolecule serves to direct formation of the native or wild-typeconformation of the opsin molecule or to stabilize a correctly foldedopsin protein, thereby facilitating insertion of the nowcorrectly-folded opsin into the membrane of a rod cell. Again, withoutwishing to be bound by theory, said insertion may help to maintain thewild-type conformation of opsin and the opsin-binding agent is free todiffuse out of the binding pocket, whereupon the pocket is available forbinding to retinal to form light-sensitive rhodopsin.

Other methods of the invention provide a means to restore photoreceptorfunction in a mammalian eye containing a mis-folded opsin protein thatcauses reduced photoreceptor function, comprising contacting saidmis-folded opsin protein with an opsin-binding agent (e.g., anon-retinoid) that reversibly binds (e.g., that binds non-covalently) ator near the retinal binding pocket. In other embodiments, binding of theopsin-binding agent to the mis-folded opsin protein competes with11-cis-retinal for binding in said binding pocket. Desirably, binding ofthe opsin-binding agent restores the native conformation of saidmis-folded opsin protein.

In preferred embodiments, the mammalian eye is a human eye. Inadditional embodiments, said contacting occurs by administering saidopsin-binding agent (e.g., non-retinoid) to a mammal afflicted with anophthalmic condition, such as a condition characterized by reducedphotoreceptor function. In various embodiments, the condition is the wetor dry form of macular degeneration, diabetic RP, a retinal or maculardystrophy, Stargardt's disease, Sorsby's dystrophy, autosomal dominantdrusen, Best's dystrophy, peripherin mutation associate with maculardystrophy, dominant form of Stargart's disease, North Carolina maculardystrophy, light toxicity (e.g., due to retinal surgery), or retinitispigmentosa. The administration may be topical administration or bysystemic administration, the latter including oral administration,intraocular injection or periocular injection. Topical administrationcan include, for example, eye drops containing an effective amount of anagent of the invention in a suitable pharmaceutical carrier.

In another embodiment, the present invention also provides a method ofstabilizing a mutant opsin protein, comprising contacting said mutantopsin protein with a non-retinoid opsin-binding agent that reversiblybinds non-covalently (for example, at or in the retinal binding pocket)to said mutant opsin protein to prevent retinoid binding in said bindingpocket, thereby stabilizing said mutant opsin protein.

The present invention also provides a method of ameliorating loss ofphotoreceptor function in a mammalian eye, comprising administering aneffective amount of an opsin-binding agent, such as a non-retinoid, to amammal afflicted with a mutant opsin protein that has reduced affinityfor 11-cis-retinal, whereby the opsin binding agent reversibly binds(e.g., non-covalently) to the retinal binding pocket of said mutantopsin, thereby ameliorating loss of photoreceptor function in saidmammalian eye. In one embodiment, the contacting occurs by administeringsaid opsin-binding agent to a mammal afflicted with said reducedphotoreceptor function, wherein said administering may be by topicaladministration or by systemic administration, the latter including oral,intraocular injection or periocular injection, and the former includingthe use of eye drops containing an agent of the invention. Such loss ofphotoreceptor function may be a partial loss or a complete loss, andwhere a partial loss it may be to any degree between 1% loss and 99%loss. In addition, suth loss may be due to the presence of a mutationthat causes mis-folding of the opsin, such as where the mutation is theP23H mutation. In another embodiment, the opsin binding agent isadministered to ameliorate an opthalmic condition related to themislocalization of an opsin protein. In one embodiment, the inventionprovides for the treatment of a subject having the dry form ofage-related macular degeneration, where at least a portion of the opsinpresent in an ocular photoreceptor cell (e.g., a rod or cone cell) ismislocalized. The mislocalized protein fails to be inserted into themembrane of a photoreceptor cell, where its function is required forvision. Administration of the opsin binding agent to a subject having amislocalized opsin protein rescues, at least in part, opsinlocalization. Accordingly, the invention is useful to prevent or treatan ophthalmic condition related to opsin mislocalization or toameliorate a symptom thereof.

The present invention provides a method for treating and/or preventingan ophthalmic condition or a symptom thereof, including but not limitedto, wet or dry form of macular degeneration, retinitis pigmentosa, aretinal or macular dystrophy, Stargardt's disease, Sorsby's dystrophy,autosomal dominant drusen, Best's dystrophy, peripherin mutationassociate with macular dystrophy, dominant form of Stargart's disease,North Carolina macular dystrophy, light toxicity (e.g., due to retinalsurgery), or retinitis pigmentosa in a subject, such as a human patient,comprising administering to a subject afflicted with, or at risk ofdeveloping, one of the aforementioned conditions or another ophthalmiccondition related to the expression of a misfolded or mislocalized opsinprotein using a therapeutically effective amount of an opsin-bindingagent, e.g., an agent that shows positive activity when tested in anyone or more of the screening assays of the invention.

Such a method may also comprise administering to said subject at leastone additional agent selected from the group consisting of a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp90 chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, anda histone deacetylase inhibitor, wherein the opsin-binding compound andthe additional compound are administered simultaneously or withinfourteen days of each other in amounts sufficient to treat the subject.

Here again the patient may comprise a mutation that affects proteinfolding where said mutation(s) causes mis-folding, e.g., in an opsinprotein, and may be any of the mutations recited elsewhere herein, suchas a P23H mutation. In other embodiments, the patient has an ophthalmiccondition that is related to the mislocalization of an opsin protein.The mislocalized opsin fails to insert into the membrane of aphotoreceptor cell (e.g., a rod or cone cell). In general, this failurein localization would effect only a portion of the opsin present in anocular cell of a patient.

In particular examples of the methods of the invention, theopsin-binding compound and the additional compound are administeredwithin ten days of each other, more preferably within five days of eachother, even more preferably within twenty-four hours of each other andmost preferably are administered simultaneously. In one example, theopsin-binding compound and the additional compound are administereddirectly to the eye. Such administration may be intra-ocular. In otherexamples, the opsin-binding compound and the additional compound areeach incorporated into a composition that provides for their long-termrelease, such as where the composition is part of a microsphere,nanosphere, or nano emulsion. In one example, the composition isadministered via a drug-delivery device that effects long-term release.Such methods also contemplate administering a vitamin A supplement alongwith an agent of the invention.

As described herein, the opsin-binding agents useful in the methods ofthe invention are available for use alone or in combination with one ormore additional compounds to treat or prevent conditions associated withthe wet or dry form of macular degeneration, retinitis pigmentosa, aretinal or macular dystrophy, Stargardt's disease, Sorsby's dystrophy,autosomal dominant drusen, Best's dystrophy, peripherin mutationassociate with macular dystrophy, dominant form of Stargart's disease,North Carolina macular dystrophy, light toxicity (e.g., due to retinalsurgery), retinitis pigmentosa or another ophthalmic condition relatedto the expression of a misfolded or mislocalized opsin protein. In oneembodiment, an opsin-hinding compound of the invention (e.g., anon-retinoid or a retinoid that fails to covalently bind to opsin) isadministered to a subject identified as having or at risk of developingsuch a condition. Optionally, the opsin binding agent is administeredtogether with another therapeutic agent. In another embodiment, anon-retinoid opsin-binding compound of the invention is used incombination with a synthetic retinoid (e.g., as disclosed in U.S. PatentPublication No. 2004-0242704), and optionally with another activecompound (e.g., as discussed herein). In still another exemplaryembodiment, an opsin-binding compound is administered in combinationwith the proteasomal inhibitor MG132, the autophagy inhibitor3-methyladenine, a lysosomal inhibitor, such as ammonium chloride, theER-Golgi transport inhibitor brefeldin A, the Hsp90 chaperone inhibitorGeldamycin, the heat shock response activator Celastrol, the glycosidaseinhibitor, and/or the histone deacetylase inhibitor Scriptaid, or anyother agent that can stabilize a mutant P23H opsin protein in abiochemically functional conformation that allows it to associate with11-cis-retinal to form rhodopsin.

In specific embodiments, an opsin-binding compound is a non-polymeric(e.g., a small molecule, such as those disclosed herein for use in themethods of the invention) compound having a molecular weight less thanabout 1000 daltons, less than 800, less than 600, less than 500, lessthan 400, or less than about 300 daltons. In certain embodiments, acompound of the invention increases the amount (e.g., from or in a cell)of a stably-folded and/or complexed mutant protein by at least 10%, 15%,20%, 25%, 50%, 75%, or 100% compared to an untreated control cell orprotein.

Proteasomal Inhibitors

The 26S proteasome is a multicatalytic protease that cleaves ubiquinatedproteins into short peptides. MG-132 is one proteasomal inhibitor thatmay be used. MG-132 is particularly useful for the treatment of lighttoxicity and other ocular diseases related to the accumulation of visualcycle products (e.g., all-trans-retinal, A2E, lipofuscin), proteinaggregation or protein misfolding. Other proteasomal inhibitors usefulin combination with of the invention in the methods of the inventioninclude lactocystin (LC), clasto-lactocystin-beta-lactone, PSI(N-carbobenzoyl-Ile-Glu-(OtBu)-Ala-Leu-CHO), MG-132(N-carbobenzoyl-Leu-Leu-Leu-CHO), MG-115(Ncarbobenzoyl-Leu-Leu-Nva-CHO), MG-101 (N-Acetyl-Leu-Leu-norLeu-CHO),ALLM (NAcetyl-Leu-Leu-Met-CHO), N-carbobenzoyl-Gly-Pro-Phe-leu-CHO,N-carbobenzoyl-Gly-Pro-Ala-Phe-CHO, N-carbobenzoyl-Leu-Leu-Phe-CHO, andsalts or analogs thereof. Other proteasomal inhibitors and their usesare described in U.S. Pat. No. 6,492,333.

Autophagy Inhibitors

Autophagy is an evolutionarily conserved mechanism for the degradationof cellular components in the cytoplasm, and serves as a cell survivalmechanism in starving cells. During autophagy pieces. of cytoplasmbecome encapsulated by cellular membranes, forming autophagic vacuolesthat eventually fuse with lysosomes to have their contents degraded.Autophagy inhibitors may be used in combination with an opsin-binding oropsin-stabilizing compound of the invention. Autophagy inhibitors usefulin combination with a of the invention in the methods of the inventioninclude, but are not limited to, 3-methyladenine, 3-methyl adenosine,adenosine, okadaic acid, N⁶-mercaptopurine riboside (N⁶-MPR), anaminothiolated adenosine analog, 5-amino-4-imidazole carboxamideriboside (AICAR), bafilomycin A1, and salts or analogs thereof.

Lysosomal Inhibitors

The lysosome is a major site of cellular protein degradation.Degradation of proteins entering the cell by receptor-mediatedendocytosis or by pinocytosis, and of plasma membrane proteins takesplace in lysosomes. Lysosomal inhibitors, such as ammonium chloride,leupeptin, trans-epoxysaccinyl-L-leucylamide-(4-guanidino) butane,L-methionine methyl ester, ammonium chloride, methylamine, chloroquine,and salts or analogs thereof, are useful in combination with anopsin-binding or opsin-stabilizing compound of the invention.

HSP90 Chaperone Inhibitors

Heat shock protein 90 (Hsp90) is responsible for chaperoning proteinsinvolved in cell signaling, proliferation and survival, and is essentialfor the conformational stability and function of a number of proteins.HSP-90 inhibitors are useful in combination with an opsin-binding oropsin-stabilizing compound in the methods of the invention. HSP-90inhibitors include benzoquinone ansamycin antibiotics, such asgeldanamycin and 17-allylamino-17-demethoxygeldanamycin (I7-AAG), whichspecifically bind to Hsp90, alter its function, and promote theproteolytic degradation of substrate proteins. Other HSP-90 inhibitorsinclude, but are not limited to, radicicol, novobiocin, and any Hsp90inhibitor that binds to the Hsp90 ATP/ADP pocket.

Heat Shock Response Activators

Celastrol, a quinone methide triterpene, activates the human heat shockresponse. In combination with an opsin-binding or opsin-stabilizingcompound in methods of the invention, celastrol and other heat shockresponse activators are useful for the treatment of PCD. Heat shockresponse activators include, but are not limited to, celastrol,celastrol methyl ester, dihydrocelastrol diacetate, celastrol butylester, dihydrocelastrol, and salts or analogs thereof.

Histone Deacetylase Inhibitors

Regulation of gene expression is mediated by several mechanisms,including the post-translational modifications of histones by dynamicacetylation and deacetylation. The enzymes responsible for reversibleacetylationldeacetylation processes are histone acetyltransferases(HATs) and histone deacetylases (HDACs), respectively. Histonedeacetylase inhibitors include Scriptaid, APHA Compound 8, Apicidin,sodium butyrate, (−)-Depudecin, Sirtinol, trichostatin A, and salts oranalogs thereof. Such inhibitors may be used in combination withcompounds of the invention in the methods disclosed herein.

Glycosedase Inhibitors

Glycosidase inhibitors are one class of compounds that are useful in themethods of the invention, when administered in combination with anopsin-binding or opsin-stabilizing compound of the invention.Castanospermine, a polyhydroxy alkaloid isolated from plant sources,inhibits enzymatic glycoside hydrolysis. Castanospermine and itsderivatives are particularly useful for the treatment of light toxicityor of an ocular Protein Conformation Disorder, such as RP. Also usefulin the methods of the invention are other glycosidase inhibitors,including australine hydrochloride, 6-Acetamido-6-deoxy-castanospermine,which is a powerful inhibitor of hexosaminidases, Deoxyfuconojirimycinhydrochloride (DFJ7), Deoxynojirimycin (DNJ), which inhibits glucosidaseI and II, Deoxygalactonojirimycin hydrochloride (DGJ), winch inhibitsα-D-galactosidase, Deoxymannojirimycin hydrochloride (DM1),2R,5R-Bis(hydroxymethyl)-3R,4R-dihydroxypyrrolidine (DMDP), also knownas 2,5-dideoxy-2,5-imino-D-mannitol, 1,4-Dideoxy-1,4-imino-D-mannitolhydrochloride, (3R,4R,5R,6R)-3,4,5,6-Tetrahydroxyazepane Hydrochloride,which inhibits b-N-acetylglucosaminidase, 1,5-Dideoxy-1,5-imino-xylitol,which inhibits β-glucosidase, and Kifunensine, an inhibitor ofmannosidase 1. Also useful in combination with an opsin-binding oropsin-stabilizing compound are N-butyldeoxynojirimycin (EDNJ), N-nonylDNJ (NDND, N-hexyl DNJ (I5TDNJ), N-methyldeoxynojirimycin (MDNJ), andother glycosidase inhibitors known in the art. Glycosidase inhibitorsare available commercially, for example, from Industrial ResearchLimited (Wellington, New Zealanti) and methods of using them aredescribed, for example, in U.S. Pat. Nos. 4,894,388, 5,043,273,5,103,008, 5,844,102, and 6,831,176; and in U.S. Patent Publication Nos.20020006909.

Pharmaceutical Compositions

The present invention features pharmaceutical preparations comprisingcompounds together with pharmaceutically acceptable carriers, where thecompounds provide for the inhibition of visual cycle products, such asall-trans-retinal or other products formed from 11-cis-retinal. Suchpreparations have both therapeutic and prophylactic applications. In oneembodiment, a pharmaceutical composition includes an opsin-binding orstabilizing compound (e.g., a compound identified using the methods ofExample 1) or a pharmaceutically acceptable salt thereof; optionally incombination with at least one additional compound that is a proteasomalinhibitor, an autophagy inhibitor, a lysosomal inhibitor, an inhibitorof protein transport from the ER to the Golgi, an Hsp9O chaperoneinhibitor, a heat shock response activator, a glycosidase inhibitor, ora histone deacetylase inhibitor. The opsin-binding or opsin-stabilizingcompound is preferably not a natural or synthetic retinoid. Theopsin-binding or opsin-stabilizing compound and the additional compoundare formulated together or separately. Compounds of the invention may beadministered as part of a pharmaceutical composition. The non-oralcompositions should be sterile and contain a therapeutically effectiveamount of the opsin-binding or opsin-stabilizing compound in a unit ofweight or volume suitable for administration to a subject. Thecompositions and combinations of the invention can be part of apharmaceutical pack, where each of the compounds is present inindividual dosage amounts.

The phrase “pharmaceutically acceptable” refers to those compounds ofthe present invention, compositions containing such compounds, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

Non-oral pharmaceutical compositions of the invention to be used forprophylactic or therapeutic administration should be sterile. Sterilityis readily accomplished by filtration through sterile filtrationmembranes (e.g., 0.2 μm membranes), by gamma irradiation, or any othersuitable means known to those skilled in the art. Therapeuticopsin-binding or opsin-stabilizing compound compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle. These compositions ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. The compounds may be combined, optionally, with apharmaceutically acceptable excipient.

The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction that wouldsubstantially impair the desired pharmaceutical efficacy.

Compounds of the present invention can be contained in apharmaceutically acceptable excipient. The exciplent preferably containsminor amounts of additives such as substances that enhance isotonicityand chemical stability. Such materials are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetate, lactate, tartrate, and otherorganic acids or their salts; tris-hydroxymethylaminomethane (TRIS),bicarbonate, carbonate, and other organic bases and their salts;antioxidants, such as ascorbic acid; low molecular weight (for example,less than about ten residues) polypeptides, e.g., polyarginine,polylysine, polyglutamate and polyaspartate; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers, such aspolyvinylpyrrolidone (PVP), polypropylene glycols (PPGs), andpolyethylene glycols (PEGs); amino acids, such as glycine, glutamicacid, aspartic acid, histidine, lysine, or arginine; monosaccharides,disaccharides, and other carbohydrates including cellulose or itsderivatives, glucose, mannose, sucrose, dextrins or sulfatedcarbohydrate derivatives, such as heparin, chondroitin sulfate ordextran sulfate; polyvalent metal ions, such as divalent metal ionsincluding calcium ions, magnesium ions and manganese ions; chelatingagents, such as ethylenediamine tetraacetic acid (EDTA); sugar alcohols,such as mannitol or sorbitol; counterions, such as sodium or ammonium;and/or nonionic surfactants, such as polysorbates or poloxamers. Otheradditives may be included, such as stabilizers, anti-microbials, inertgases, fluid and nutrient replenishers (i.e., Ringer's dextrose),electrolyte replenishers, which can be present in conventional amounts.

The compositions, as described above, can be administered in effectiveamounts. The effective amount will depend upon the mode oradministration, the particular condition being treated and the desiredoutcome. It may also depend upon the stage of the condition, the age andphysical condition of the subject, the nature of concurrent therapy, ifany, and like factors well known to the medical practitioner. Fortherapeutic applications, it is that amount sufficient to achieve amedically desirable result.

With respect to a subject suffering from, or at risk of developing,light toxicity, such as that due to ocular surgery, an effective amountis an amount sufficient to reduce the rate or extent of formation andaccumulation of visual cycle products, such as all-trans-retinal, orlipofuscin, or A2E as well as preventing photocell apoptosis as a resultof excessive rhodopsin activation. Here, the compounds of the presentinvention would be from about 0.01 mg/kg per day to about 1000 mg/kg perday. It is expected that doses ranging from about 50 to about 2000 mg/kgwill be suitable. Lower doses will result from certain forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day are contemplated to achieve appropriatesystemic levels of a composition of the present invention.

A variety of administration routes are available. The methods of theinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. In one preferred embodiment, acomposition of the invention is administered intraocularly. Other modesof administration include oral, rectal, topical, intraocular, buccal,intravaginal, intracistemal, intracerebroventricular, intratracheal,nasal, transdermal, within/on implants, or parenteral routes.Compositions comprising a composition of the invention can be added to aphysiological fluid, such as to the intravitreal humor. For CNSadministration, a variety of techniques are available for promotingtransfer of the therapeutic across the blood brain barrier includingdisruption by surgery or injection, drugs which transiently openadhesion contact between the CNS vasculature endothelial cells, andcompounds that facilitate translocation through such cells. Oraladministration can be preferred for prophylactic treatment because ofthe convenience to the patient as well as the dosing schedule.

Pharmaceutical compositions of the invention can optionally furthercontain one or more additional proteins as desired, including plasmaproteins, proteases, and other biological material, so long as it doesnot cause adverse effects upon administration to a subject. Suitableproteins or biological material may be obtained from human or mammalianplasma by any of the purification methods known and available to thoseskilled in the art; from supernatants, extracts, or lysates ofrecombinant tissue culture, viruses, yeast, bacteria, or the like thatcontain a gene that expresses a human or mammalian plasma protein whichhas been introduced according to standard recombinant DNA techniques; orfrom the fluids (e.g., blood, milk, lymph, urine or the like) ortransgenic animals that contain a gene that expresses a human plasmaprotein which has been introduced according to standard transgenictechniques.

Pharmaceutical compositions of the invention can comprise one or more pHbuffering compounds to maintain the pH of the formulation at apredetermined level that reflects physiological pH, such as in the rangeof about 5.0 to about 8.0 (e.g., 6.0, 6.5, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.8). The pH buffering compound used in the aqueousliquid formulation can be an amino acid or mixture of amino acids, suchas histidine or a mixture of amino acids such as histidine and glycine.Alternatively, the pH buffering compound is preferably an agent whichmaintains the pH of the formulation at a predetermined level, such as inthe range of about 5.0 to about 8.0, and which does not chelate calciumions. Illustrative examples of such pH buffering compounds include, butare not limited to, imidazole and acetate ions. The pH bufferingcompound may be present in any amount suitable to maintain the pH of theformulation at a predetermined level.

Pharmaceutical compositions of the invention can also contain one ormore osmotic modulating agents, i.e., a compound that modulates theosmotic properties (e.g., tonicity, osmolality and/or osmotic pressure)of the formulation to a level that is acceptable to the blood stream andblood cells of recipient individuals. The osmotic modulating agent canbe an agent that does not chelate calcium ions. The osmotic modulatingagent can be any compound known or available to those skilled in the artthat modulates the osmotic properties of the formulation. One skilled inthe art may empirically determine the suitability of a given osmoticmodulating agent for use in the inventive formulation. Illustrativeexamples of suitable types of osmotic modulating agents include, but arenot limited to: salts, such as sodium chloride and sodium acetate;sugars, such as sucrose, dextrose, and mannitol; amino acids, such asglycine; and mixtures of one or more of these agents and/or types ofagents. The osmotic modulating agent(s) maybe present in anyconcentration sufficient to modulate the osmotic properties of theformulation.

Compositions comprising an opsin-binding or opsin-stabilizing compoundof the present invention can contain multivalent metal ions, such ascalcium ions, magnesium ions and/or manganese ions. Any multivalentmetal ion that helps stabilize the composition and that will notadversely affect recipient individuals may be used. The skilled artisan,based on these two criteria, can determine suitable metal ionsempirically and suitable sources of such metal ions are known, andinclude inorganic and organic salts.

Pharmaceutical compositions of the invention can also be a non-aqueousliquid formulation. Any suitable non-aqueous liquid may be employed,provided that it provides stability to the active agents (a) containedtherein. Preferably, the non-aqueous liquid is a hydrophilic liquid.Illustrative examples of suitable non-aqueous liquids include: glycerol;dimethyl sulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols,such as ethylene glycol, diethylene glycol, triethylene glycol,polyethylene glycol (“PEG”) 200, PEG 300, and PEG 400; and propyleneglycols, such as dipropylene glycol, tripropylene glycol, polypropyleneglycol (“PPG”) 425, PPG 725, PPG 1000, PEG 2000, PEG 3000 and PEG 4000.

Pharmaceutical compositions of the invention can also be a mixedaqueous/non-aqueous liquid formulation. Any suitable non-aqueous liquidformulation, such as those described above, can be employed along withany aqueous liquid formulation, such as those described above, providedthat the mixed aqueous/non-aqueous liquid formulation provides stabilityto the compound contained therein. Preferably, the non-aqueous liquid insuch a formulation is a hydrophilic liquid. Illustrative examples ofsuitable non-aqueous liquids include: glycerol; DMSO; EMS; ethyleneglycols, such as PEG 200, PEG 300, and PEG 400; and propylene glycols,such as PPG 425, PPG 725, PEG 1000, PEG 2000, PEG 3000 and PEG 4000.Suitable stable formulations can permit storage of the active agents ina frozen or an unfrozen liquid state. Stable liquid formulations can bestored at a temperature of at least −70° C., but can also be stored athigher temperatures of at least 0° C., or between about 0′C and about42° C., depending on the properties of the composition. It is generallyknown to the skilled artisan that proteins and polypeptides aresensitive to changes in pH, temperature, and a multiplicity of otherfactors that may affect therapeutic efficacy.

In certain embodiments a desirable route of administration can be bypulmonary aerosol. Techniques for preparing aerosol delivery systemscontaining polypeptides are well known to those of skill in the art.Generally, such systems should utilize components that will notsignificantly impair the biological properties of the antibodies, suchas the paratope binding capacity (see, for example, Sciarra and Cutie,“Aerosols,” in Remington's Pharmaceutical Sciences 18th edition, 1990,pp 1694-1712; incorporated by reference). Those of skill in the art canreadily modify the various parameters and conditions for producingpolypeptide aerosols without resorting to undue experimentation.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of compositions of the invention, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as polylactides (U.S. Pat. No.3,773,919; European Patent No. 58,481), poly(lactide-glycolide),copolyoxalates polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acids, such as poly-D-(−)-3-hydroxybutyric acid(European Patent No. 133,988), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, K R. et at, Biopolymers 22: 547-556),poly (2-hydroxyethyl methacrylate) or ethylene vinyl acetate (Langer, etal., J. Biomed. Mater. Res. 15:267-277; Langer, B. Chem. Tech.12:98-105), and polyanhydrides.

Other examples of sustained-release compositions include semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Delivery systems also include non-polymer systems thatare: lipids including sterols such as cholesterol, cholesterol estersand fatty acids or neutral fats such as mono-, di- and tri-glycerides;hydrogel release systems such as biologically-derived bioresorbablehydrogel (i.e., chitin hydrogels or chitosan hydrogels); sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially filled implants; and thelike. Specific examples include, but are not limited to: (a) aerosionalsystems in which the agent is contained in a form within a matrix suchas those described in 13.5. U.S. Pat. Nos. 4,452,775, 4,667,014,4,748,034 and 5,239,660 and (b) diffusional systems in which an activecomponent permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,832,253, and 3,854,480.

Another type of delivery system that can be used with the methods andcompositions of the invention is a colloidal dispersion system.Colloidal dispersion systems include lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Liposomes are artificial membrane vessels, which are useful as adelivery vector in vivo or in vitro. Large unilamellar vessels (LUV),which range in size from 0.2-4.0 μm, can encapsulate largemacromolecules within the aqueous interior and be delivered to cells ina biologically active form (Fraley, R., and Papahadjopoulos, D., TrendsBiochem. Sci. 6: 77-80).

Liposomes can be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Liposomes are commercially available from GibcoBRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed ofcationic lipids such as N-[1-(2, 3dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications, for example, in DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. (USA) 82:3688-3692 (1985); K. Hwang et al., Proc. Natl, Acad.Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Liposomes also have beenreviewed by Gregoriadis, G., Trends Biotechnol., 3: 235-241.

Another type of vehicle is a biocompatible microparticle or implant thatis suitable for implantation into the mammalian recipient. Exemplarybioerodible implants that are useful in accordance with this method aredescribed in PCT International application no. PCTIUS/03307 (PublicationNo. WO 95/24929, entitled “Polymeric Gene Delivery System”). PCT/US/0307describes biocompatible, preferably biodegradable polymeric matrices forcontaining an exogenous gene under the control of an appropriatepromoter. The polymeric matrices can be used to achieve sustainedrelease of the exogenous gene or gene product in the subject.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein an agent is dispersed throughout a solidpolymeric matrix) or a microcapsule (wherein an agent is stored in thecore of a polymeric shell). Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Other forms of the polymeric matrix for containing an agent includefilms, coatings, gels, implants, and stents. The size and composition ofthe polymeric matrix device is selected to result in favorable releasekinetics in the tissue into which the matrix is introduced. The size ofthe polymeric matrix further is selected according to the method ofdelivery that is to be used. Preferably, when an aerosol route is usedthe polymeric matrix and composition are encompassed in a surfactantvehicle. The polymeric matrix composition can be selected to have bothfavorable degradation rates and also to be formed of a material, whichis a bioadhesive, to further increase the effectiveness of transfer. Thematrix composition also can be selected not to degrade, but rather torelease by diffusion over an extended period of time. The deliverysystem can also be a biocompatible microsphere that is suitable forlocal, site-specific delivery. Such microspheres are disclosed inChickering, D. B., et al., Biotechnot. Bioeng, 52: 96-101; Mathiowitz,B., et at., Nature 386: 410-414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the compositions of the invention to the subject. Suchpolymers may be natural or synthetic polymers. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

Exemplary synthetic polymers which can be used to form the biodegradabledelivery system include: polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates,polyvinyl, alcohols, polyvinyl ethers, polyvinyl esters, polyvinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluoses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose proplonate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulfate sodium salt,poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate),poly(isodecyl mcthacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene, poly(ethylene glycol), poly(ethylene oxide),poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate),poly(vinyl chloride), polystyrene, poly(viny Ipyrrolidone), and polymersof lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone),and natural polymers such as alginate and other polysaccharidesincluding dextran and cellulose, collagen, chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), albumin and other hydrophilicproteins, zein and other prolamines and hydrophobic proteins, copolymersand mixtures thereof. In general, these materials degrade either byenzymatic hydrolysis or exposure to water in vivo, by surface or bulkerosion.

Methods of Ocular Delivery

The compositions of the invention are particularly suitable for treatingocular diseases or conditions, such as light toxicity, in particularlight toxicity related to an ocular surgical procedure.

In one approach, the compositions of the invention are administeredthrough an ocular device suitable for direct implantation into thevitreous of the eye. The compositions of the invention may be providedin sustained release compositions, such as those described in, forexample, U.S. Pat. Nos. 5,672,659 and 5,595,760. Such devices are foundto provide sustained controlled release of various compositions to treatthe eye without risk of detrimental local and systemic side effects. Anobject of the present ocular method of delivery is to maximize theamount of drug contained in an intraocular device or implant whileminimizing its size in order to prolong the duration of the implant.See, e.g., U.S. Pat. Nos. 5,378,475; 6,375,972, and 6,756,058 and U.S.Publications 20050096290 and 200501269448. Such implants may bebiodegradable and/or biocompatible implants, or may be non-biodegradableimplants.

Biodegradable ocular implants are described, for example, in U.S. PatentPublication No. 20050048099. The implants may be permeable orimpermeable to the active agent, and may be inserted into a chamber ofthe eye, such as the anterior or posterior chambers or may be implantedin the sclera, transchoroidal space, or an avascularized region exteriorto the vitreous. Alternatively, a contact lens that acts as a depot forcompositions of the invention may also be used for drug delivery.

In a preferred embodiment, the implant may be positioned over anavascular region, such as on the sclera, so as to allow for transcleraldiffusion of the drug to the desired site of treatment, e.g. theintraocular space and macula of the eye. Furthermore, the site oftranscleral diffusion is preferably in proximity to the macula. Examplesof implants for delivery of a composition of the invention include, butare not limited to, the devices described in U.S. Pat. Nos. 3,416,530;3,828,777; 4,014,335; 4,300,557; 4,327,725; 4,853,224; 4,946,450;4,997,652; 5,147,647; 164,188; 5,178,635; 5,300,114; 5,322,691;5,403,901; 5,443,505; 5,466,466; 5,476,511; 5,516,522; 5,632,984;5,679,666; 5,710,165; 5,725,493; 5,743,274; 5,766,242; 5,766,619;5,770,592; 5,773,019; 5,824,072; 5,824,073; 5,830,173; 5,836,935;5,869,079, 5,902,598; 5,904,144; 5,916,584; 6,001,386; 6,074,661;6,110,485; 6,126,687; 6,146.366; 6,251,090; and 6,299,895, and in WO01/30323 and WO 01/28474, all of which are incorporated herein byreference.

Examples include, but are not limited to the following: a sustainedrelease drug delivery system comprising an inner reservoir comprising aneffective amount of an agent effective in obtaining a desired local orsystemic physiological or pharmacological effect, an inner tubeimpermeable to the passage of the agent, the inner tube having first andsecond ends and covering at least a portion of the inner reservoir, theinner tube sized and formed of a material so that the inner tube iscapable of supporting its own weight, an impermeable member positionedat the inner tube first end, the impermeable member preventing passageof the agent out of the reservoir through the inner tube first end, anda permeable member positioned at the inner tube second end, thepermeable member allowing diffusion of the agent out of the reservoirthrough the inner tube second end; a method for administering a compoundof the invention to a segment of an eye, the method comprising the stepof implanting a sustained release device to deliver the compound of theinvention to the vitreous of the eye or an implantable, sustainedrelease device for administering a compound of the invention to asegment of an eye; a sustained release drug delivery device comprising:a) a drug core comprising a therapeutically effective amount of at leastone first agent effective in obtaining a diagnostic effect or effectivein obtaining a desired local or systemic physiological orpharmacological effect; b) at least one unitary cup essentiallyimpermeable to the passage of the agent that surrounds and defines aninternal compartment to accept the drug core, the unitary cup comprisingan open top end with at least one recessed groove around at least someportion of the open top end of the unitary cup; c) a permeable plugwhich is permeable to the passage of the agent, the permeable plug ispositioned at the open top end of the unitary cup wherein the grooveinteracts with the permeable plug holding it in position and closing theopen top end, the permeable plug allowing passage of the agent out ofthe drug core, though the permeable plug, and out the open top end ofthe unitary cup; and d) at least one second agent effective in obtaininga diagnostic effect or effective in obtaining a desired local orsystemic physiological or pharmacological effect; or a sustained releasedrug delivery device comprising: an inner core comprising an effectiveamount of an agent having a desired solubility and a polymer coatinglayer, the polymer layer being permeable to the agent, wherein thepolymer coating layer completely covers the inner core.

Other approaches for ocular delivery include the use of liposomes totarget a compound of the present invention to the eye, and preferably toretinal pigment epithelial cells and/or Bruch's membrane. For example,the compound maybe complexed with liposomes in the manner describedabove, and this compound/liposome complex injected into patients with anophthalmic condition, such as light toxicity, using intravenousinjection to direct the compound to the desired ocular tissue or cell.Directly injecting the liposome complex into the proximity of theretinal pigment epithelial cells or Bruch's membrane can also providefor targeting of the complex with some forms of ocular PCD. In aspecific embodiment, the compound is administered via intra-ocularsustained delivery (such as VITRASERT or ENVISION. In a specificembodiment, the compound is delivered by posterior subtenons injection.In another specific embodiment, microemulsion particles containing thecompositions of the invention are delivered to ocular tissue to take uplipid from Bruchs membrane, retinal pigment epithelial cells, or both.

Nanoparticles are a colloidal carrier system that has been shown toimprove the efficacy of the encapsulated drug by prolonging the serumhalf-life. Polyalkylcyanoacrylates (PACAs) nanoparticles are a polymercolloidal drug delivery system that is in clinical development, asdescribed by Stella et al, J. Pharm. Sci., 2000. 89: p. 1452-1464;Brigger et al., Tnt. J. Pharm., 2001. 214: p. 37-42; Calvo et al.,Pharm. Res., 2001. 18: p. 1157-1166; and Li et al., Biol. Pharm. Bull.,2001. 24: p. 662-665. Biodegradable poly (hydroxyl acid₆), such as thecopolymers of poly (lactic acid) (PLA) and poly (lactic-co-glycolide)(PLGA) are being extensively used in biomedical applications and havereceived FDA approval for certain clinical applications. In addition,PEG-PLGA nanoparticles have many desirable carrier features including(i) that the agent to be encapsulated comprises a reasonably high weightfraction (loading) of the total carrier system; (ii) that the amount ofagent used in the first step of the encapsulation process isincorporated into the final carrier (entrapment efficiency) at areasonably high level; (iii) that the carrier have the ability to befreeze-dried and reconstituted in solution without aggregation; (iv)that the carrier be biodegradable; (v) that the carrier system be ofsmall size; and (vi) that the carrier enhance the particles persistence.

Nanoparticles are synthesized using virtually any biodegradable shellknown in the art. In one embodiment, a polymer, such as poly(lactic-acid) (PLA) or poly (lactic-co-glycolic acid) (PLGA) is used.Such polymers are biocompatible and biodegradable, and are subject tomodifications that desirably increase the photochemical efficacy andcirculation lifetime of the nanoparticle. In one embodiment, the polymeris modified with a terminal carboxylic acid group (COOH) that increasesthe negative charge of the particle and thus limits the interaction withnegatively charge nucleic acid aptamers. Nanoparticles are also modifiedwith polyethylene glycol (PEG), which also increases the half-life andstability of the particles in circulation. Alternatively, the COOH groupis converted to an N-hydroxysuccinimide (NHS) ester for covalentconjugation to amine-modified aptamers.

Biocompatible polymers useful in the composition and methods of theinvention include, but are not limited to, polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, poly(viny|pyrrolidone), polyglycolides,polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose,hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic esters, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, cellulosesulfate sodium salt poly-methyl methacrylate), poly(ethyl methacrylate),poly(butyl methacrylate), poly(isobutyl methacrylate poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate),poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate),polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide),poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl acetate,polyvinyl chloride polystyrene, poly(vinyl pyrrolidone), polyhyaluronicacids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid,alginate, chitosan, poly(methyl methacrylates), poly(ethylmethacrylates), poly(butyl methacrylate), poly(isobutyl methacrylate),poly(hexyl methacrylate) poly(isodecyl methaerylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylatee), poly(isobutyl acrylate), poly(octadecylacrylate) and combinations of any of these, In one embodiment, thenanoparticles of the invention include PEG-PLGA polymers.

Compositions of the invention may also be delivered topically. Fortopical delivery, the compositions are provided in any pharmaceuticallyacceptable excipient that is approved for ocular delivery. Preferably,the composition is delivered in drop form to the surface of the eye. Forsome application, the delivery of the composition relies on thediffusion of the compounds through the comea to the interior of the eye.

Those of skill in the art will recognize that treatment regimens forusing the compounds of the present invention to treat light toxicity orother opthalmic conditions (e.g., RP) can be straightforwardlydetermined. This is not a question of experimentation, but rather one ofoptimization, which is routinely conducted in the medical arts. In vivostudies in nude mice often provide a starting point from which to beginto optimize the dosage and delivery regimes. The frequency of injectionwill initially be once a week, as has been done in some mice studies.However, this frequency might be optimally adjusted from one day toevery two weeks to monthly, depending upon the results obtained frontthe initial clinical trials and the needs of a particular patient.

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. For certain embodiments it is envisioned that the dosage mayvary from between about 1 mg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other embodiments this dose maybe about 1, 5, 10, 25, 50, 75,100, 150, 10 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, 5000 mg/Kg body weight. in other embodiments, it is envisaged thatlower does may be used, such doses may be in the range of about 5 mgcompound/Kg body to about 20 mg compound/Kg body. In other embodimentsthe doses may be about 8, 10, 12, 14, 16 15 or 18 mg/Kg body weight. Ofcourse, this dosage amount may be adjusted upward or downward, as isroutinely done in such treatment protocols, depending on the results ofthe initial clinical trials and the needs of a particular patient.

Screening Assays

Useful compounds of the invention are compounds of the formual (I) thatreversibly bind to a native or mutated opsin protein, such as in or nearthe 11-cis-retinal binding pocket. The non bleachable or slowlybleachable pigment rhodopsins formed from these small molecule opsinbindings will prevent light toxicity related to, for example, theaccumulation of visual cycle products as well as apoptotic photocelldeath resulting from excessive rhodopsin stimulation. Such binding willcommonly inhibit, if not prevent, binding of retinoids, especially11-cis-retinal, to the binding pocket and thereby reduce formation ofvisual cycle products, such as all-trans-retinal. Any number of methodsare available for carrying out screening assays to identify suchcompounds. In one approach, an opsin protein is contacted with acandidate compound or test compound that is a non-retinoid in thepresence of 11-cis-retinal or retinoid analog and the rate or yield offormation of chromophore is determined. If desired, the binding of thenon-retinoid to opsin is characterized. Preferably, the non-retinoidbinding to opsin is non-covalent and reversible. Thus, inhibition ofrhodopsin formation by a non-retinoid indicates identification of asuccessful test compound. An increase in the amount of rhodopsin isassayed, for example, by measuring the protein's absorption at acharacteristic wavelength (e.g., 498 nm for rhodopsin) or by measuringan increase in the biological activity of the protein using any standardmethod (e.g., enzymatic activity association with a liganti). Usefulcompounds inhibit binding of 11-cis-retinal (and formation of rhodopsin)by at least about 10%, 15%, or 20%, or preferably by 25%, 50%, or 75%,or most preferably by up to 90% or even 100%.

The efficacy of the identified compound is assayed in an animal modelshowing the effects of light toxicity. For example, the efficacy ofcompounds disclosed herein have been demonstrated using transgenic micethat contain a mutant elaov 4 gene important in fatty acid synthesis andtransgenic mice that produce a mutant ABCR protein that affects howall-trans-retinal is shuttled. The amount of lipofuscin produced in suchmice was determined using compounds of the invention and shown to beproduced at a reduced rate resulting in slower accumulation of toxicvisual cycle products. In either case, the cellular phenotype is thesame and lipofuscin is accumulated at an accelerated rate whensuccessful test compounds are not administered.

Alternatively, the efficacy of compounds useful in the methods of theinvention may be determined by exposure of a mammalian eye to a highintensity light source prior to, during, or following administration ofa test compound, followed by determination of the amount of visual cycleproducts (e.g., all-trans retinal, A2E, or lipofuscin) formed as aresult of exposure to the high intensity light source, wherein acompound of the invention will have reduced the amount of visual cycleproducts related to the exposure.

In sum, preferred test compounds identified by the screening methods ofthe invention are non-retinoids, are selective for opsin and bind in areversible, non-covalent manner to opsin protein. In addition, theiradministration to transgenic animals otherwise producing increasedlipofuscin results in a reduced rate of production or a reducedaccumulation of lipofuscin in the eye of said animal. Compoundsidentified according to the methods of the invention are useful for thetreatment of light toxicity or other ophthalmic condition in a subject,such as a human patient.

Combination Therapies

Compositions of the invention useful for the prevention of lighttoxicity, as well as AMD and retinitis pigmentosa, can optionally becombined with additional therapies as heretofore described.

EXAMPLES

The following non-limiting examples further describe and enable one ofordinary skill in the art to make use of the invention.

Example 1 (E)-3-(2,6,6-Trimethylcyclohex-1-enyl)acrylic acid

The title compound, obtained as a colorless crystalline solid (14.2 g,52%), was prepared from β-ionone (26.7 g, 0.139 mol) according to theprocedure of [Shimasaki, H.; Kagechika, H.; Fukasawa, H.; Kawachi, E.;Shudo, K. Chem. Pharm. Bull. 1995, 43, 100-107]. R_(f)=0.4 (25:75 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 12.16 (br s, 1H), 7.56 (d,J=16.0 Hz, 1H), 5.85 (d, J=16.0 Hz, 1H), 2.08 (t, J=6.0 Hz, 2H), 1.79(s, 3H), 1.66-1.58 (m, 2H), 1.50-1.46 (m, 2H), 1.08 (s, 6H) ppm.

Example 2 (E)-3-(2,6,6-Trimethylcyclohex-1-enyl)acrylamide 2a(E)-3-(2,6,6-Trimethycyclohex-1-enyl)acryloyl chloride

To a round bottom flask charged with(E)-3-(2,6,6-trimethylcyclohex-1-enyl) acrylic acid (1, 6.00 g, 3.00mmol) in anhydrous dichloromethane (2.5 mL) under argon was added oxalylchloride (0.50 mL, 5.50 mmol) dropwise via syringe. To this stirredsolution was added two drops of N,N-dimethylformamide (DMF) and thereaction was stirred at room temperature for 3 hours. The reactionmixture was concentrated in vacuo (40° C.) to yield a yellow-brown oilwhich was carried forward without further purification.

2b (E)-3-(2,6,6-Trimethycyclohex-1-enyl)acrylamide

In a round bottom flask (E)-3-(2,6,6-trimethylcyclohex-1-enyl)acryloylchloride (320 mg, 1.50 mmol) was dissolved in tetrahydrofuran (THF, 6.0mL). The reaction mixture was cooled to 0° C. and a solution of ammoniumhydroxide (0.4 mL) was added. The reaction mixture was warmed to roomtemperature while stirred for 4 hours. The crude reaction mixture wasconcentrated in vacuo (35° C.) and purified by preparative plate thinlayer chromatography (5:95 methanol:chloroform) to afford a yellowamorphous solid (153 mg, 53%). R_(f)=0.60 (3:97 methanol:chloroform);¹H-NMR (400 MHz, CDCl₃) δ 7.37 (d, J=15.5 Hz, 1H), 6.42 (s, 1H), 5.83(d, J=15.5 Hz, 1H), 5.61 (s, 1H), 2.09-2.04 (m, 2H), 1.76 (s, 3H),1.65-1.62 (m, 2H), 1.50-1.48 (m, 2H), 1.07 (s, 6H); Mass spectrum(ESI+ve) m/z 193.9 (MH⁺).

Example 3 (E)-N-Methyl-3-(2,6,6-trimethylcyclohex-1-enyl)acrylamide

To a solution of (E)-3-(2,6,6-trimethyicyclohex-1-enyl) acrylic acid (1,50.0 mg, 0.257 mmol) in DMF (1.0 mL) was added2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 97.7 mg, 0.257 mmol). The solution wasstirred at room temperature for 30 minutes then diisopropylethylamine(66.4 mg, 0.514 mmol) and methylamine hydrochloride (17.4 mg, 0.257mmol) was added to the reaction mixture. The reaction was then stirredat room temperature for 4 hours.

The reaction was quenched with a 1M solution of hydrochloric acid (2 mL)and the biphasic mixture was separated. The organic layer wasconcentrated in vacuo (40° C.) and the crude material loaded on tosilica gel for purification via flash column chromatography running anisocratic eluent of 30% ethyl acetate in hexanes. The title compound wasisolated as a white solid (56 mg, 86%). Mp=84-88° C.; R_(f)=0.34 (50:50ethyl acetate:hexanes); ¹H NMR (400 MHz, CDCl₃) 7.30 (d, J=15.5, 1H),5.74 (d, J=15.5 Hz, 1H), 5.48 (d, J=1.5 Hz, 1H), 2.93 (d, J=5.0 Hz, 3H),2.05 (t, J=6.0 Hz, 2H), 1.74 (s, 3H), 1.65-1.60 (m, 2H), 1.49 (dd,J=7.5, 4.0 Hz, 2H), 1.06 (s, 6H); Mass spectrum (ESI+ve) m/z 208 (MH⁺).

Example 4 (E)-N,N-Dimethyl-3-(2,6,6-trimethylcyclohex-1-enyl)acrylamide

The title compound, obtained as an colorless oil (18 mg, 32%), wasprepared from the product of Example 1 by following the procedure ofExample 3 except dimethylamine (2.0 M solution in tetrahydrofuran) wassubstituted for methylamine hydrochloride. R_(f)=0.44 (50:50 ethylacetate: hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.34 (d, J=15.6 Hz, 1H),6.25 (d, J=15.5 Hz, 1H), 3.10 (s, 3H), 3.05 (s, 3H), 2.05 (t, J=6.0 Hz,2H), 1.77 (s, 3H), 1.64 (dd, J=8.0, 4.0 Hz, 2H), 1.51-1.47 (m, 2H), 1.07(s, 6H); Mass spectrum (ESI+ve) m/z 221 (MH⁺).

Example 5(E)-1-(Piperidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as an colorless oil (62 mg, 95%), wasprepared from the product of Example 1 by following the procedure ofExample 3 except piperidine was substituted for methylaminehydrochloride.

R_(f)=0.50 (40:60 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.30(d, J=13.5 Hz, 1H), 6.24 (d, J=15.5 Hz, 1H), 3.70-3.45 (m, 4H), 2.04 (t,J=6.0 Hz, 2H), 1.75 (s, 3H), 1.70-1.56 (m, 8H), 1.48 (dd, J=7.5, 4.0 Hz,2H), 1.06 (s, 6H); Mass spectrum (ESI+ve) m/z 262 (MH⁺).

Example 6(E)-1-Morpholino-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as an colorless oil (60.0 mg, 89%), wasprepared from the product of Example 1 by following the procedure ofExample 3 except morpholine was substituted for methylaminehydrochloride and O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) was substituted for2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU). R_(f)=0.40 (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.36 (d, J=15.5 Hz, 1H), 6.20 (d, J=17.0 Hz,1H), 3.77-3.47 (m, 7H), 3.10-3.02 (m, 1H), 2.03 (d, J=5.5 Hz, 2H), 1.74(s, 3H), 1.61 (dd, J=7.5, 4.0 Hz, 2H), 1.51-1.42 (m, 2H), 1.05 (s, 6H);Mass spectrum (ESI+ve) m/z 264 (MH⁺).

Example 7(E)-1-(4-Methyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one 7a (E)-tert-Butyl4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxylate

The title compound, obtained as an colorless oil (193 mg, 98%), wasprepared from the product of Example 1 by following the procedure ofExample 3 except tert-butyl 1,4-diazepane-1-carboxylate was substitutedfor methylamine hydrochloride andO-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)was substituted for2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (HATU). R_(f)=0.40 (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.47-7.30 (m, 1H), 6.18 (d, J=15.5 Hz, 1H),3.73-3.31 (m, 8H), 2.04 (s, 2H), 1.95-1.83 (m, 2H), 1.75 (d, J=12.0 Hz,3H), 1.61 (dd, J=12.0, 6.0 Hz, 2H), 1.45 (m, 11H), 1.06 (s, 6H); Massspectrum (ESI+ve) m/z 377 (MH⁺).

7b(E)-1-(1,4-Diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

To a solution of the product of Example 7a (160 mg, 0.425 mmol) indichloromethane (3 mL) was added dropwise a 2.0 M solution ofhydrochloric acid in diethyl ether (0.43 mL, 0.85 mmol). The reactionmixture was stirred at room temperature for 18 hours. The title compoundwas isolated by flash column chromatography using a 10-25% methanol indichloromethane solvent gradient to yield a dark yellow oil (115 mg,87%). R_(f)=0.30 (90:10 dichloromethane:methanol); ¹H NMR (400 MHz,CDCl₃) δ 7.78-7.23 (m, 2H), 6.40-6.01 (m, 2H), 4.32-4.13 (m, 1H), 3.81(m, 4H), 3.22 (m, 4H), 2.12 (m, 3H), 1.84-1.25 (m, 9H), 1.08 (m, 5H),0.92 (m, 3H); Mass spectrum (ESI+ve) m/z 277 (MH⁺).

7c(E)-1-(4-Methyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

To a solution of(E)-1-(1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one(7b, 50.0 mg, 0.181 mmol) and potassium carbonate (50.0 mg, 0.362 mmol)were dissolved in dichloromethane (3 mL) at room temperature. To thisstirred solution was added iodomethane (25.7 mg, 0.181 mmol) dropwisevia syringe. The reaction mixture was stirred for 18 hours at roomtemperature.

The reaction mixture was filtered to remove the excess potassiumcarbonate and then concentrated in vacuo (40° C.) to yield a crudesolid. The crude material was purified using flash columnchromatography. (98:2 dichloromethane:methanol) to provide a pale yellowoil (16.0 mg, 31%). R_(f)=0.75 (90:9:1 dichloromethane:methanol:ammoniumhydroxide); ¹H NMR (400 MHz, CDCl₃) δ 7.34 (dd, J=15.5, 7.5 Hz, 1H),6.18 (dd, J=15.5, 6.0 Hz, 1H), 3.80-3.57 (m, 4H), 2.74-2.50 (m, 4H),2.38 (d, J=8.5 Hz, 3H), 2.07-1.89 (m, 4H), 1.73 (s, 3H), 1.61 (td,J=12.5, 6.0 Hz, 2H), 1.50-1.41 (m, 2H), 1.04 (s, 6H); Mass spectrum(ESI+ve) m/z 291 (MH⁺).

Example 8(E)-1-(4-Ethyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as yellow oil (37 mg, 34%), was preparedfrom the product of Example 7b by following the procedure of Example 7cexcept ethyliodide was substituted for iodomethane. R_(f)=0.23 in (5:95methanol:chloroform); ¹H NMR (400 MHz, CDCl₃) δ 7.34 (m, 1H), 6.19 (d,J=15.5 Hz, 1H), 3.82-3.67 (m, 2H), 3.68-3.57 (m, 2H), 2.81-2.50 (m, 6H),2.02 (m, 2H), 1.93 (m, 2H), 1.74 (s, 3H), 1.67-1.56 (m, 2H), 1.53-1.41(m, 2H), 1.25 (s, 1H), 1.13-0.98 (m, 8H); Mass spectrum (ESI+ve) m/z 305(MH⁺).

Example 9(E)-1-(4-propyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as yellow oil (12 mg, 21%), was preparedfrom the product of Example 7b by following the procedure of Example 7cexcept propyl bromide was substituted with iodomethane. R_(f)=0.23 in(5:95 methanol:chloroform); ¹H NMR (400 MHz, CDCl₃) δ 7.32 (dd, J=15.5,7.5 Hz, 1H), 6.16 (m, 1H), 3.76-3.63 (m, 2H), 3.58 (t, J=5.0 Hz, 2H),2.78-2.66 (m, 2H), 2.67-2.56 (m, 3H), 2.51-2.36 (m, 2H), 2.00 (t, J=6.0Hz, 2H), 1.87 (m, 3H), 1.71 (s, 3H), 1.59 (m, 2H), 1.52-1.40 (m, 4H),1.02 (s, 6H), 0.85 (m, 3H); Mass spectrum (ESI+ve) m/z 319 (MH⁺).

Example 10(E)-1-(4-Acetyl-1,4-diazepan-1-yl)-3-(2,6,6-trmethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a colorless oil (40.4 mg, 71%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except acetyl chloride was substituted for iodomethane.R_(f)=0.80 (90:10 dichloromethane:methanol); ¹H NMR (400 MHz, CDCl₃) δ7.47-7.27 (m, 1H), 6.15 (d, J=15.5 Hz, 1H), 3.58 (m, 9H), 2.77 (s, 1H),2.11-1.96 (m, 5H), 1.97-1.77 (m, 3H), 1.69 (d, J=16.5 Hz, 3H), 1.58 (dd,J=7.5, 4.0 Hz, 2H), 1.48-1.39 (m, 2H), 1.00 (s, 6H); Mass spectrum(ESI+ve) m/z 319 (MH⁺).

Example 11(E)-1-(4-Propionyl-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a colorless oil (56.1 mg, 93%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except propionyl chloride was substituted for iodomethane.R_(f)=0.25 (75:25 dichloromethane:ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 7.37 (q, J=15.5 Hz, 1H), 6.17 (dd, J=15.5, 6.0 Hz, 1H),3.76-3.44 (m, 8H), 2.38-2.27 (m, 2H), 2.02 (s, 2H), 1.94-1.82 (m, 2H),1.73 (s, 3H), 1.59 (d, J=6.0 Hz, 2H), 1.50-1.41 (m, 2H), 1.14 (td,J=14.5, 7.5 Hz, 3H), 1.03 (s, 6H); Mass spectrum (ESI+ve) m/z 333 (MH⁺).

Example 12(E)-1-(4-(2,2,2-Trifluoroacetyl)-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a colorless oil (40.5 mg, 60%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except trifluoroacetic anhydride was substituted foriodomethane. R_(f)=0.75 (75:25 dichloro-methane:ethyl acetate); ¹H NMR(400 MHz, CDCl₃) δ 7.41 (m, 1H), 6.17 (dd, J=15.5, 6.5 Hz, 1H), 3.64 (m,8H), 2.09-1.91 (m, 4H), 1.74 (s, 3H), 1.68-1.56 (m, 2H), 1.48 (dd,J=7.5, 4.0 Hz, 2H), 1.05 (s, 6H); Mass spectrum (ESI+ve) m/z 373 (MH⁺).

Example 13(E)-4-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide

The title compound, obtained as yellow oil (20 mg, 21%), was preparedfrom the product of Example 7b by following the procedure of Example 7cexcept trimethylsilyl isocyanate was substituted for iodomethane.R_(f)=0.57 (100% ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 7.39 (m, 1H),6.18 (d, J=15.0 Hz, 1H), 5.19-4.66 (m, 2H), 3.84-3.32 (m, 8H), 2.02 (m,2H), 1.73 (s, 3H), 1.60 (m, 2H), 1.46 (m, 2H), 1.25 (m, 2H), 1.04 (s,6H); Mass spectrum (ESI+ve) m/z 320 (MH⁺).

Example 14(E)-N-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide

(E)-1-(1,4-Diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one(7b, 50.0 mg, 0.181 mmol) and potassium carbonate (100 mg, 0.362 mmol)were dissolved in anhydrous dichloromethane (5 mL) under argon at roomtemperature. To this stirred solution was added 4-nitrophenylchloroformate (73.0 mg, 0.362 mmol). The reaction mixture was stirredfor 18 hours at room temperature.

The reaction mixture was transferred to a microwave vial and the solventwas removed in vacuo. To the residue was added methylamine (56.2 mg,1.81 mmol) and the sealed microwave vial was heated at 100° C. for 30minutes in a microwave reactor. The contents of the reaction vial werethen poured into 10 mL of water, extracted with dichloromethane (3×10mL) and the combined organic layers were dried over sodium sulfate. Thesolvent was removed in vacuo (40° C.) to yield a white solid. The crudematerial was purified using flash column chromatography (98:2dichloromethane:methanol) to provide a colorless oil (31 mg, 0.092 mmol,25%). R_(f)=0.23 (98:2 dichloromethane:methanol); ¹H NMR (400 MHz,CDCl₃) δ 7.37 (dd, J=15.5, 15.5 Hz, 1H), 6.18 (dd, J=15.5, 6.0 Hz, 1H),4.48-4.38 (m, 1H), 3.76-3.25 (m, 8H), 2.81 (s, 3H), 2.08-1.82 (m, 4H),1.74 (s, 3H), 1.68-1.60 (m, 4H), 1.48-1.42 (m, 2H), 1.05 (s, 6H); Massspectrum (ESI+ve) m/z 334 (MH⁺).

Example 15(E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide

The title compound, obtained as colorless oil (63 mg, 100%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except ethyl isocyanate was substituted for iodomethane.R_(f)=0.1 (80:20 ethyl acetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.40(t, J=15.5 Hz, 1H), 6.21 (dd, J=15.5, 6.5 Hz, 1H), 4.38 (m, 1H), 3.71(d, 2H), 3.54 (m, 4H), 3.34-3.24 (m, 2H), 2.83 (s, 2H), 2.06 (t, J=6.0Hz, 2H), 2.03-1.96 (m, 1H), 1.90 (t, J=6.0 Hz, 1H), 1.7 (s, 3H), 1.64(m, 2H), 1.52-1.46 (m, 2H), 1.16 (t, J=7.0 Hz, 3H), 1.07 (s, 6H); Massspectrum (ESI+ve) m/z 348 (MH⁺).

Example 16(E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide

The title compound, obtained as a pale yellow oil (61.0 mg, 93%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except propyl isocyanate was substituted for iodomethane.R_(f)=0.15 (50:50 ethyl acetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.37(t, J=15.5 Hz, 1H), 6.18 (dd, J=15.5, 7.0 Hz, 1H), 4.51-4.33 (m, 2H),3.74 (t, J=5.5 Hz, 1H), 3.62 (t, J=5.5 Hz, 2H), 3.57-3.44 (m, 4H), 3.34(t, J=6.0 Hz, 1H), 3.15 (m, 3H), 2.08-1.93 (m, 3H), 1.86 (dd, J=12.0,6.0 Hz, 1H), 1.74 (s, 4H), 1.67-1.42 (m, 7H), 1.04 (s, 6H), 0.91 (t,J=7.5 Hz, 5H); Mass spectrum (ESI+ve) m/z 350.3 (MH⁺).

Example 17(E)-N-Isopropyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxamide

The title compound, obtained as a pale yellow oil (66.0 mg, 99%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except isopropyl isocyanate was substituted for iodomethane.R_(f)=0.70 (90:10 dichloro-methane:methanol); ¹H NMR (400 MHz, CDCl₃) δ7.36 (t, J=16.0 Hz, 1H), 6.18 (dd, J=15.5, 6.5 Hz, 1H), 4.29 (m, 1H),3.95 (dd, J=12.5, 6.0 Hz, 1H), 3.58 (m, 7H), 3.33 (s, 1H), 2.79 (d,J=2.5 Hz, 1H), 2.11-1.78 (m, 4H), 1.73 (s, 3H), 1.61 (dd, J=5.5, 3.5 Hz,2H), 1.52-1.38 (m, 3H), 1.12 (m, 6H), 1.03 (s, 6H); Mass spectrum(ESI+ve) m/z 362 (MH⁺).

Example 18(E)-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxylate

The title compound, obtained as a colorless solid (40.1 mg, 67%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except methyl chloroformate was substituted for iodomethane.R_(f)=0.45 (75:25 dichloromethane:ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 7.36 (t, J=17.0 Hz, 1H), 6.15 (t, J=12.5 Hz, 1H), 3.75-3.34 (m,11H), 2.02 (t, J=6.0 Hz, 2H), 1.86 (m, 3H), 1.73 (s, 3H), 1.60 (m, 2H),1.51-1.38 (m, 2H), 1.03 (s, 6H); Mass spectrum (ESI+ve) m/z 335 (MH⁺).

Example 19(E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carboxylate

The title compound, obtained as pale yellow oil (61 mg, 98%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except ethyl chloroformate was substituted for iodomethane.R_(f)=0.1 (25:75 ethyl acetate:dichloromethane); ¹H NMR (400 MHz, CDCl₃)δ 7.34 (d, 1H), 6.16 (dd, J=15.5, 6.5 Hz, 1H), 4.52 (s, 1H), 3.75-3.41(m, 7H), 3.37-3.15 (m, 3H), 2.05-1.78 (m, 4H), 1.71 (s, 3H), 1.63-1.53(m, 2H), 1.48-1.39 (m, 2H), 1.10 (t, J=7.0 Hz, 3H), 1.01 (s, 6H); Massspectrum (ESI+ve) m/z 349 (MH⁺).

Example 20(E)-14-(Methylsulfonyl)-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as colorless oil (48 mg, 75%), was preparedfrom the product of Example 7b by following the procedure of Example 7cexcept methanesulfonyl chloride was substituted for iodomethane.R_(f)=0.7 (10:90 methanol:dichloromethane); ¹H NMR (400 MHz, CDCl₃) δ7.41 (m, 1H), 6.25-6.11 (m, 1H), 3.75 (m, 4H), 3.41 (m, 4H), 2.84 (s,3H), 1.98 (m, 2H), 1.74 (s, 3H), 1.65-1.54 (m, 2H), 1.51-1.39 (m, 4H),1.09-0.98 (s, 6H); Mass spectrum (ESI+ve) m/z 355 (MH⁺).

Example 21(E)-1-(4-(Ethylsulfonyl)-1,4-diazepan-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as pale yellow oil (58 mg, 86%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except ethanesulfonyl chloride was substituted foriodomethane. R_(f)=0.75 (10:90 methanol:dichloromethane); ¹H NMR (400MHz, CDCl₃) δ 7.40 (m, 1H), 6.18 (m, 1H), 3.72 (m, 5H), 3.53-3.30 (m,2H), 2.99 (m, 2H), 2.78 (s, 2H), 2.08-1.89 (m, 4H), 1.73 (s, 3H), 1.45(m, 2H), 1.31 (t, J=7.5 Hz, 2H), 1.21 (t, J=7.5 Hz, 2H), 1.03 (s, 6H);Mass spectrum (ESI+ve) m/z 369 (MH⁺).

Example 22(E)-1-(4-(Trifluoromethylsulfonyl)-1,4-diazepan-1-yl)-3-(2,6,6-tri-methylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as colorless oil (22 mg, 30%), was preparedfrom the product of Example 7b by following the procedure of Example 7cexcept trifluoromethanesulfonyl chloride was substituted foriodomethane. R_(f)=0.25 (25:75 ethyl acetate:dichloromethane); ¹H NMR(400 MHz, CDCl₃) δ 7.45 (m, 1H), 6.17 (m, 1H), 3.67 (m, 8H), 2.09-1.95(m, 4H), 1.74 (s, 3H), 1.61 (m, 2H), 1.53-1.44 (m, 2H), 1.05 (s, 6H);Mass spectrum (ESI+ve) m/z 409 (MH⁺).

Example 23(E)-N-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothioamide

The title compound, obtained as pale yellow oil (50 mg, 79%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except methyl thioisocyanate was substituted for iodomethane.R_(f)=0.15 (25:75 ethyl acetate:dichloromethane); ¹H NMR (400 MHz,CDCl₃) δ 7.35 (t, J=16.0 Hz, 1H), 6.19 (m, 1H), 5.87 (m, 1H), 4.17 (t,J=5.0 Hz, 1H), 3.98-3.72 (m, 5H), 3.57 (m, 3H), 3.12 (d, J=4.0 Hz, 3H),2.78 (s, 2H), 1.98 (m, 4H), 1.73 (s, 3H), 1.64-1.55 (m, 2H), 1.46 (m,2H), 1.03 (s, 6H); Mass spectrum (ESI+ve) m/z 350 (MH⁺).

Example 24(E)-N-Ethyl-43-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothioamide

The title compound, obtained as pale yellow oil (66 mg, 100%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except ethyl thioisocyanate was substituted for iodomethane.R_(f)=0.28 (80:20 ethyl acetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.43(t, J=15.5 Hz, 1H), 6.23 (m, 1H), 5.48 (s, 1H), 4.20 (t, J=5.0 Hz, 1H),4.02-3.90 (m, 2H), 3.82-3.76 (m, 1H), 3.71 (m, 2H), 3.60 (m, 3H), 2.83(s, 2H), 2.07 (m, 2H), 2.03-1.94 (m, 1H), 1.77 (s, 3H), 1.69-1.58 (m,4H), 1.54-1.47 (m, 2H), 1.26 (t, J=7.0 Hz, 3H), 1.08 (s, 6H); Massspectrum (ESI+ve) m/z 364 (MH⁺).

Example 25(E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothioamide

The title compound, obtained as pale yellow oil (64 mg, 94%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except propyl thioisocyanate was substituted for iodomethane.R_(f)=0.55 (25:75 ethyl acetate:dichloromethane); ¹H NMR (400 MHz,CDCl₃) δ 7.38 (t, J=16.5 Hz, 1H), 6.21 (m, 1H), 5.67 (m, 1H), 4.18 (m,1H), 3.99-3.74 (m, 4H), 3.65-3.50 (m, 5H), 2.09-1.93 (m, 4H), 1.75 (s,3H), 1.68-1.56 (m, 4H), 1.48 (m, 2H), 1.05 (s, 6H), 0.95 (t, J=7.5 Hz,3H); Mass spectrum (ESI+ve) m/z 378 (MH⁺).

Example 26(E)-N-Isopropyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)-1,4-diazepane-1-carbothloamide

The title compound, obtained as pale yellow oil (64 mg, 94%), wasprepared from the product of Example 7b by following the procedure ofExample 7c except isopropyl thioisocyanate was substituted foriodomethane. R_(f)=0.5 (25:75 ethyl acetate:dichloromethane); ¹H NMR(400 MHz, CDCl₃) δ 7.38 (t, J=16.5 Hz, 1H), 6.21 (m, 1H), 5.34 (m, 7.5Hz, 1H), 4.64 (m, 1H), 4.13 (m, 1H), 3.83 (m, 4H), 3.57 (m, 3H),2.08-1.92 (m, 4H), 1.75 (s, 3H), 1.62 (m, 2H), 1.51-1.43 (m, 2H), 1.24(d, J=6.5 Hz, 6H), 1.05 (s, 6H); Mass spectrum (ESI+ve) m/z 378 (MH⁺).

Example 27(E)-1-(4-Methylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a yellow oil (112 mg, 81%) was preparedfrom the product of Example 1 by following the procedure of Example 6except N-methylpiperazine was substituted for morpholine. R_(f)=0.28(90:10 dichloromethane:methanol); ¹H NMR (400 MHz, CD₂Cl₂) δ 7.26 (d,1H, J=16 Hz), 6.25 (d, 1H, J=16 Hz), 3.74-3.47 (m, 4H), 2.41-2.38 (m,4H), 2.30 (s, 3H), 2.08 (t, 2H, J=6.4 Hz), 1.77 (s, 3H), 1.69-1.63 (m,2H), 1.53-1.50 (m, 2H), 1.08 (s, 6H); Mass spectrum (ESI+ve) m/z 277.2(MH⁺).

Example 28(E)-1-(4-Ethylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one28a (E)-tert-Butyl 4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a yellow oil (3.60 g, 99%), was preparedfrom the product of Example 1 by following the procedure of Example 3except tert-butyl piperazine-1-carboxylate was substituted formethylamine hydrochloride andO-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)was substituted for2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate (HATU). R_(f)=0.21 in (20:80 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.38 (d, J=15.5 Hz, 1H),6.21 (d, J=13.0 Hz, 1H), 3.70-3.48 (m, 8H), 2.06 (t, J=6.0 Hz, 2H), 1.77(s, 3H), 1.67-1.62 (m, 2H), 1.49 (s, 11H), 1.10 (s, 6H); Mass spectrum(ESI+ve) m/z 363 (MH⁺).

28b(E)-1-(Piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-nyl)prop-2-en-1-one

The title compound, obtained as a yellow oil (250 mg, 96%) was preparedfrom the product of Example 28a by following the procedure of example7b. R^(f)=0.36 (91:8:1 dichloromethane:methanol:ammonium hydroxide); ¹HNMR (400 MHz, CD₂Cl₂) δ 7.25 (d, J=16.0 Hz, 1H), 6.24 (d, J=16.0 Hz,1H), 3.70-3.45 (m, 4H), 2.88-2.83 (m, 4H), 2.07 (t, J=6.0 Hz, 2H), 1.77(s, 3H), 1.69-1.63 (m, 2H), 1.53-1.50 (m, 2H), 1.08 (s, 6H); Massspectrum (ESI+ve) m/z 263 (MH⁺).

28c(E)-1-(4-Ethylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a yellow oil (21 mg, 38%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept ethyl bromide was substituted for iodomethane, acetonitrile wassubstituted for dichloromethane and the reaction was heated at 50° C.instead of room temperature. R_(f)=0.28 (95:5 dichloromethane:methanol);¹H NMR (400 MHz, CDCl₃) δ 7.30 (d, J=16.0 Hz, 1H), 6.23 (d, J=16.0 Hz,1H), 3.80-3.60 (m, 4H), 2.55-2.42 (m, 6H), 2.07 (t, J=6.5 Hz, 2H), 1.78(s, 3H), 1.69-1.62 (m, 2H), 1.53-1.50 (m, 2H), 1.12 (t, J=6.0 Hz, 3H),1.07 (s, 6H); Mass spectrum (ESI+ve) m/z 291 (MH⁺).

Example 29(E)-1-(4-Propylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a clear oil (3.5 mg, 6%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept propyl iodide was substituted for iodomethane. R_(f)=0.53 (90:10dichloromethane:methanol); ¹H NMR (400 MHz, CDCl₃) δ 7.35 (d, J=16.0 Hz,1H), 6.22 (d, J=16.0 Hz, 1H), 3.79-3.46 (m, 4H), 2.95-2.84 (m, 4H), 2.05(t, J=6.0 Hz, 2H), 1.76 (s, 3H), 1.71 (s, 2H), 1.63 (m, 2H), 1.53-1.45(m, 2H), 1.07 (s, 6H); Mass spectrum (ESI+ve) m/z 305 (MH⁺).

Example 30(E)-1-(4-Acetylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a clear oil (52 mg, 90%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept that acetyl chloride was substituted for iodomethane. R_(f)=0.16(98:2 dichloromethane:methanol); ¹H NMR (400 MHz, CD₂Cl₂) δ 7.32 (d,J=16.0 Hz, 1H), 6.25 (d, J=16.0 Hz, 1H), 3.79-3.46 (m, 8H), 2.14-2.03(m, 5H), 1.78 (s, 3H), 1.70-1.61 (m, 2H), 1.55-1.50 (m, 2H), 1.09 (s,6H); Mass spectrum (ESI+ve) m/z 305 (MH⁺).

Example 31(E)-1-(4-Propionylpiperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a clear oil (31 mg, 51%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept that propionyl chloride was substituted for iodomethane.R_(f)=0.10 (98:2 dichloromethane:methanol); ¹H NMR (400 MHz, CDCl₃) δ7.40 (d, J=16.0 Hz, 1H), 6.22 (d, J=16.0 Hz, 1H), 3.81-3.47 (m, 8H),2.40 (dt, J=7.5, 5.0 Hz, 4H), 2.07 (t, J=6.0 Hz, 2H), 1.77 (s, 3H), 1.64(m, 2H), 1.53-1.46 (m, 2H), 1.23-1.14 (m, 2H), 1.08 (s, 6H); Massspectrum (ESI+ve) m/z 319 (MH⁺).

Example 32(E)-1-(4-(2,2,2-Trifluoroacetyl)piperazin-1-yl)-3-(2,6,6-trimethylcyclo-hex-1-enyl)prop-2-en-1-one

The title compound, obtained as a clear oil (65 mg, 96%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept that trifluoroacetic anhydride was substituted for iodomethane.R_(f)=0.25 (98:2 dichloro-methane:methanol); ¹H NMR (400 MHz, CDCl₃) δ7.42 (d, J=16.0 Hz, 1H), 6.20 (d, J=16.0 Hz, 1H), 3.70 (m, 8H),2.10-2.01 (m, 2H), 1.77 (s, 3H), 1.64 (m 2H), 1.53-1.45 (m, 2H), 1.07(s, 6H); Mass spectrum (ESI+ve) m/z 359 (MH⁺).

Example 33(E)-4-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

The product of Example 28b (50 mg, 0.189 mmol) and triethylamine (79 μL,0.567 mmol) were dissolved in anhydrous dichloromethane (4 mL) at roomtemperature under argon and stirred for 5 minutes. To this stirredreaction mixture was added trimethylsilyl isocyanate (76 μL, 0.567mmol). The reaction mixture was stirred at room temperature for 18hours.

The reaction mixture was poured into a saturated aqueous solution ofammonium chloride (10 mL) and was extracted with dichloromethane (3×10mL). The combined organic phases were dried over sodium sulfate and theconcentrated in vacuo. The product was purified by flash columnchromatography to yield the title compound as a white solid (60 mg,quantitative). Mp=144° C.; R_(f)=0.31 (96:4 dichloromethane:methanol);¹H NMR (300 MHz, CDCl₃) δ 7.39 (d, J=16.0 Hz, 1H), 6.21 (d, J=16.0 Hz,1H), 4.61-4.60 (m, 2H), 3.90-3.40 (m, 8H), 2.06 (t, J=6.0 Hz, 2H), 1.77(s, 3H), 1.66-1.62 (m, 2H), 1.51-1.47 (m, 2H), 1.07 (s, 6H); Massspectrum (ESI+ve) m/z 306 (MH⁺).

Example 34(E)-N-Methyl-4-(3-(2,6,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a clear oil (51 mg, 85%) was preparedfrom the product of Example 28b following the procedure of Example 14.R_(f)=0.50 (50:50 ethyl acetate:hexane); ¹H NMR (400 MHz, CDCl₃) δ 7.42(d, 1H, J=16.0 Hz), 6.20 (d, 1H, J=16.0 Hz), 4.45 (br s, 1H), 3.80-3.30(m, 8H), 2.88 (s, 3H), 2.09 (t, 2H, J=6.4 Hz)), 1.77 (s, 3H), 1.70-1.64(m, 2H), 1.53-1.45 (m, 2H), 1.07 (s, 6H); Mass spectrum (ESI+ve) m/z 320(MH⁺).

Example 35(E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a white solid (25 mg, 40%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept that ethyl isocyanate was substituted for iodomethane.Mp=125-128° C.; R_(f)=0.50 (2:1 ethyl acetate:dichloromethane); ¹H NMR(400 MHz, CDCl₃) δ 7.41 (d, J=16.0 Hz, 1H), 6.22 (d, J=16.0 Hz, 1H),4.40 (br s, 1H), 3.82-3.30 (m, 10H), 2.13 (t, J=6.0 Hz, 2H), 1.77 (s,3H), 1.70-1.64 (m 2H), 1.53-1.45 (m, 2H), 1.18 (t, J=6.0 Hz, 3H), 1.08(s, 6H); Mass spectrum (ESI+ve) m/z 334 (MH⁺).

Example 36(E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a clear oil (42 mg, 57%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept that n-propyl isocyanate was substituted for iodomethane.R_(f)=0.21 (98:2 dichloromethane:methanol); ¹H NMR (400 MHz, CDCl₃) δ7.39 (d, J=16.0 Hz, 1H), 6.21 (d, J=16.0 Hz, 1H), 4.48 (s, 1H),3.84-3.33 (m, 8H), 3.24 (q, J=6.0 Hz, 2H), 2.06 (t, J=6.0 Hz, 2H), 1.77(s, 3H), 1.68-1.46 (m, 9H), 1.07 (s, 6H), 0.95 (t, J=7.5 Hz, 3H); Massspectrum (ESI+ve) m/z 348 (MH⁺).

Example 37(E)-N-Isopropyl-4-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a white solid (62 mg, 85%) was preparedfrom the product of Example 28b following the procedure of Example 7cexcept that isopropyl isocyanate was substituted for iodomethane.Mp=158-159° C.; R_(f)=0.32 (98:2 dichloro-methane:methanol); ¹H NMR (400MHz, CDCl₃) δ 7.37 (d, J=16.0 Hz, 1H), 6.20 (d, J=16.0 Hz, 1H), 4.30 (d,J=7.0 Hz, 1H), 3.99 (m, 1H), 3.82-3.31 (m, 9H), 2.05 (t, J=6.0 Hz, 2H),1.76 (s, 3H), 1.67-1.59 (m, 2H), 1.49 (m, 2H), 1.17 (d, J=6.5 Hz, 6H),1.06 (s, 6H); Mass spectrum (ESI+ve) m/z 348 (MH⁺).

Example 38 (E)-Methyl 4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a colorless oil (34.0 mg, 55%), wasprepared from the product of example 28b by following the procedure ofExample 7c except methyl chloroformate was substituted for iodomethane.R, =0.25 in (20:80 ethyl acetate:dichloromethane); ¹H-NMR (400 MHz,CDCl₃) δ 7.31 (d, J=15.5 Hz, 1H), 6.23 (d, J=15.5 Hz, 1H), 3.80-3.48 (m,8H), 2.12-2.01 (m, 2H), 1.76 (s, 3H), 1.73-1.56 (m, 5H), 1.54-1.50 (m,2H), 1.08 (s, 6H); Mass spectrum (ESI+ve) m/z 321 (MH⁺).

Example 39 (E)-Ethyl 4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a colorless oil (58.0 mg, 91%), wasprepared from the product of example 28b by following the procedure ofExample 7c except ethyl chloroformate was substituted for iodomethane.R_(f)=0.12 in (20:80 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ7.48 (d, J=15.5 Hz, 1H), 6.21 (d, J=15.5 Hz, 1H), 4.11-4.38 (m, 2H),3.78-3.51 (m, 8H), 2.08 (m, 2H), 2.07 (s, 3H), 1.72-1.61 (m, 2H),1.56-1.49 (m, 2H), 1.32 (m, 3H) 1.09 (s, 6H); Mass spectrum (ESI+ve) m/z335 (MH⁺).

Example 40(E)-1-(4-(Methylsulfonyl)piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a white solid (38.2 mg, 59%), wasprepared from the product of example 28b by following the procedure ofExample 7c except methylsulfonyl chloride was substituted foriodomethane. Mp=121-123° C.; R_(f)=0.39 in (1:99 methanol:hexanes);¹H-NMR (400 MHz, CD₂Cl₂) δ 7.35 (d, J=12.0 Hz, 1H), 6.25 (d, J=12.0 Hz,1H), 3.75 (br m, 4H), 3.25 (m, 4H), 2.78 (s, 3H), 2.08-2.01 (m, 2H),1.85 (s, 3H), 1.65-1.60 (m, 2H), 1.50-1.45 (m, 2H), 1.05 (s, 6H); Massspectrum (ESI+ve) m/z 341 (MH⁺).

Example 41(E)-1-(4-(Ethylsulfonyl)piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a white solid (35.0 mg, 52%), wasprepared from the product of example 28b by following the procedure ofExample 7c except ethylsulfonyl chloride was substituted foriodomethane. Mp=125-127° C.; R_(f)=0.15 in (2:98 methanol:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.42 (d, J=12.0 Hz, 1H), 6.22 (d, J=12.0 Hz,1H), 3.85-3.70 (m, 4H), 3.40-3.30 (m, 4H), 2.99 (q, J=6.0 Hz, 2H),2.08-2.01 (m, 2H), 1.70-1.65 (m 2H), 1.60 (s, 3H), 1.55-1.50 (m, 2H),1.40 (t, J=6.0 Hz, 3H), 1.06 (s, 6H); Mass spectrum (ESI+ve) m/z 355(MH⁺).

Example 42(E)-1-(4-(Trifluoromethylalfonyulfonyl)piperazin-1-yl)-3-(2,6,6-trimethyl-cyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a colorless oil (2.50 mg, 2.8%), wasprepared from the product of example 28b by following the procedure ofExample 7c except trifluoromethylsulfonyl chloride was substituted foriodomethane. R_(f)=0.75 in (20:80 ethyl acetate:dichloromethane); ¹H-NMR(400 MHz, CDCl₃) δ 7.45 (d, J=15.5 Hz, 1H), 6.22 (d, J=15.5 Hz, 1H),3.99-3.42 (m, 8H), 2.15 (s, 3H), 1.74-1.58 (m, 2H), 1.56-1.48 (m, 2H),1.25-1.31 (m, 2H), 1.09 (s, 6H); Mass spectrum (ESI+ve) m/z 395 (MH⁺).

Example 43(E)-N-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide

The title compound, obtained as a white solid (38.2 mg, 61%), wasprepared from the product of example 28b by following the procedure ofExample 7c except methyl isothiocyanate was substituted for iodomethane.Mp=55-57° C.; R_(f)=0.37 in (65:35 ethyl acetate:dichloromethane);¹H-NMR (400 MHz, CDCl₃) δ 7.44 (d, J=15.5 Hz, 1H), 6.22 (d, J=15.5 Hz,1H), 5.98 (s, 1H), 4.18 (s, 2H), 3.89-3.68 (m, 6H), 3.19 (s, 3H),2.12-2.08 (m, 2H), 1.75 (s, 3H), 1.72-1.61 (m, 2H) 1.52-1.49 (m, 2H),1.10 (s, 6H); Mass spectrum (ESI+ve) m/z 336 (MH⁺).

Example 44(E)-N-Ethyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide

The title compound, obtained as a white solid (42.0 mg, 63%), wasprepared from the product of example 28b by following the procedure ofExample 7c except ethyl isothiocyanate was substituted for iodomethane.Mp=133-135° C.; R_(f)=0.33 in (50:50 ethyl acetate:dichloromethane);¹H-NMR (400 MHz, CDCl₃) δ 7.42 (d, J=12.0 Hz, 1H), 6.20 (d, J=12.0 Hz,1H), 5.50 (br s, 1H), 4.25-4.10 (m, 2H), 3.95-3.65 (m 8H), 2.12-2.08 (m,2H), 1.75 (s, 3H), 1.68-1.58 (m, 2H) 1.55-1.49 (m, 2H), 1.30 (t, J=6.0Hz, 1H), 1.08 (s, 6H); Mass spectrum (ESI+ve) m/z 350 (MH⁺).

Example 45(E)-N-Propyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide

The title compound, obtained as a white solid (37.9 mg, 55%), wasprepared from the product of example 28b by following the procedure ofExample 7c except propyl isothiocyanate was substituted for iodomethane.Mp=128-130° C.; R_(f)=0.53 in (50:50 ethyl acetate:dichloromethane);¹H-NMR (400 MHz, CDCl₃) δ 7.46 (d, J=15.5 Hz, 1H), 6.23 (d, J=15.5 Hz,1H), 5.59 μl (s, 1H), 4.19 (s, 2H), 3.91-3.64 (m, 8H), 2.09 (s, 2H),1.86-1.62 (m, 7H), 1.49 (s, 2H), 1.13-0.93 (m, 9H); Mass spectrum(ESI+ve) m/z 364 (MH⁺).

Example 46(E)-N-Isopropyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carbothioamide

The title compound, obtained as a white solid (35.2 mg, 51%), wasprepared from the product of example 28b by following the procedure ofExample 7c except isopropyl isothiocyanate was substituted foriodomethane. Mp=147-148° C.; R_(f)=0.51 in (50:50 ethylacetate:dichloromethane); ¹H-NMR (400 MHz, CDCl₃) δ 7.48 (d, J=15.5 Hz,1H), 6.22 (d, J=15.5 Hz, 1H), 5.25 (s, 1H), 4.77-4.62 (m, 1H), 4.18 (s,1H), 3.92-3.63 (m, 6H), 2.18-2.06 (m, 2H), 1.78 (s, 3H), 1.71-1.63 (m,3H) 1.48-1.50 (m, 2H), 1.29 (m, 6H), 1.08 (s, 6H); Mass spectrum(ESI+ve) m/z 364 (MH⁺).

Example 47(S,E)-1-(3-Hydroxypyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The title compound, obtained as a clear oil (196 mg, 97%) was preparedfrom the product of Example 1 following the procedure of Example 3except (S)-pyrrolidin-3-ol was substituted for methylamine hydrochlorideand acetonitrile was substituted for dichloromethane. R_(f)=0.20 (ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 7.36 (m, 1H), 6.08 (dd, J=15.5, 8.5,1H), 4.54 (m, 1H), 3.76-3.58 (m, 4H), 3.03-2.88 (m, 1H), 2.05 (m, 4H),1.76 (s, 3H), 1.63 (m, 2H), 1.49 (m, 2H), 1.07 (s, 6H); Mass spectrum(ESI+ve) m/z 264 (MH⁺).

Example 48(S,E)-1-(3-(2,6,6-Trimethylcyclohex-1-enyl)acryloyl)pyrrolidin-3-ylcarbamate

Trichloroacetyl isocyanate (63 μL, 0.53 mmol) was added to a solution ofthe product of Example 47 (70 mg, 0.26 mmol). The solution was stirredovernight at room temperature and then quenched by treatment with water(0.5 mL). Ethyl acetate (20 mL) was added to dilute the reactionmixture, and the organic phase was extracted with water (20 mL). Theorganic layer was separated and dried over magnesium sulfate and thesolvent was removed in vacuo. The desired product was isolated bypreparative plate thin layer chromatography (100% EtOAc) to yield aclear oil (23 mg, 15%). R_(f)=0.42 (ethyl acetate); ¹H NMR (400 MHz,CDCl₃) δ 7.40 (d, J=15.0, 1H), 6.80 (dd, J=15.5, 1H), 5.30 (m, 1H), 4.74(m, 2H), 3.72-3.66 (m, 4H), 3.62 (m, 2H), 2.17 (m, 2H), 2.06 (s, 3H),1.77 (d, J=5.5, 2H), 1.63 (m, 2H), 1.08 (s, 6H); Mass spectrum (ESI+ve)m/z 307 (MH⁺).

Example 49(E)-1-(3-Aminopyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-onehydrochloride 49a (E)-tert-Butyl1-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl) pyrrolidin-3-yl carbamate

The title compound, obtained as white solid (210 mg, 75%), was preparedfrom the product of Example 1 following the procedure of Example 3except tert-butyl pyrrolidin-3-ylcarbamate was substituted formethylamine hydrochloride. The crude product was carries forward withoutfurther purification. R_(f)=0.2 in (40:60 ethyl acetate:hexanes); Massspectrum (ESI+ve) m/z 363 (MH⁺).

49b(E)-1-(3-Aminopyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

The product of Example 49a was dissolved in a 4N solution ofhydrochloric acid in 1,4-dioxane (2 mL) and stirred at room temperaturefor 1 hour. The reaction solution was concentrated in vacuo toapproximately 0.5 mL. The crude product was added via pipette to diethylether (50 mL) where a white precipitate was formed. The precipitate wasfiltered, washed with ether and dried under reduced pressure to yieldthe desired product as a white solid (70 mg, 84%). Mp=188° C.; ¹H NMR(400 MHz, DMSO-d) δ 8.40-8.35 (m, 3H), 7.17 (m, 1H), 6.15 (m, 1H), 3.79(m, 2H), 3.62 (m, 2H), 2.26 (m, 1H), 2.04 (m, 4H), 1.73 (s, 3H), 1.58(m, 2H), 1.45 (m, 2H), 1.03 (s, 6H); Mass spectrum (ESI+ve) m/z 263(MH⁺).

Example 50 (E)-1-(1-(3-(2,6,6-Trimethylcyclohex-1-nyl)acryloyl)pyrrolidin-3-yl) urea

The title compound, obtained as colorless oil (6.7 mg, 9%), was preparedfrom the product of Example 49b by following the procedure of Example48. R_(f)=0.25 (90:10 chloroform:methanol); ¹H NMR (400 MHz, CDCl₃) δ7.35 (d, J=15.5, 1H), 6.50 (m, 1H), 6.08 (t, J=15.5, 1H), 5.04 (br s,2H), 4.32 (m, 1H), 3.61 (m, 4H), 2.06-2.05 (m, 4H), 1.76 (s, 3H), 1.62(m, 2H), 1.48 (m, 2H), 1.07 (m, 6H); Mass spectrum (ESI+ve) m/z 306(MH⁺).

Example 514-(3-(2,6,6-Trimethylcyclohex-1-enyl)propanoyl)piperazine-1-carboxamide51a 1-(Piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-enyl)propan-1-one

Example 28b (100 mg, 0.381 mmol) was dissolved in anhydrous methanol (10mL) under argon and stirred at room temperature. To this stirredreaction mixture was added magnesium turnings (83.0 mg, 3.41 mmol). Thereaction mixture was stirred under argon at room temperature for 48hours.

Methanol was removed in vacuo and the residue was taken up in water (20mL). The aqueous phase was extracted with chloroform (3×10 mL) and thecombined organic phases were dried over sodium sulfate. The solvent wasremoved in vacuo to provide a yellow oil (62 mg crude). The product waspurified by preparative thin layer chromatography to yield the titlecompound, as clear oil (4.2 mg, 4%). R_(f)=0.39 (95:5dichloromethane:methanol); ¹H NMR (400 MHz, CDCl₃) δ 3.66-3.60 (m, 2H),3.50-3.43 (m, 2H), 2.88 (d, J=4.5 Hz, 4H), 2.36 (s, 3H), 1.93 (t, J=6.0Hz, 2H), 1.64-1.55 (m, 5H), 1.44 (dd, J=7.5, 4.0 Hz, 2H), 1.29 (m, 1H),1.17-1.10 (m, 1H), 1.02 (s, 6H); Mass spectrum (ESI+ve) m/z 265 (MH⁺).

51b4-(3-(2,6,6-Trimethylcyclohex-1-enyl)propanoyl)piperazine-1-carboxamide

The title compound, obtained as a white solid (3.0 mg, 61%) was preparedfrom the product of Example 51a following the procedure of Example 33.R_(f)=0.45 (95:5 dichloromethane:methanol); ¹H NMR (400 MHz, CDCl₃) δ4.53 (s, 2H), 3.74-3.66 (m, 2H), 3.52 (s, 4H), 3.40 (s, 2H), 2.38 (s,3H), 1.93 (t, J=6.0 Hz, 2H), 1.67-1.55 (m, 6H), 1.48-1.41 (m, 2H),1.33-1.26 (m, 2H), 1.14 (m, 2H), 1.02 (s, 6H); Mass spectrum (ESI+ve)m/z 308 (MH⁺).

Example 52(S,E)-2-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piper-azine-1-carboxamide52a (S)-tert-Butyl 4-carbamoyl-3-methylpiperazine-1-carboxylate

The title compound, obtained as a yellow foam (480 mg, quant.) wasprepared from (S)-1-Boc-3-methylpiperazine following the procedure ofExample 33. [α]_(D)=14° (c=0.005, EtOH); R_(f)=0.52 (95:5dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H NMR (400MHz, CDCl₃) δ 4.81 (s, 2H), 3.97 (m, 5H), 3.09 (m, 3H), 2.99-2.73 (m,1H), 1.47 (s, 9H), 1.42-1.36 (m, 1H), 1.18 (d, J=6.0 Hz, 3H); Massspectrum (ESI+ve) m/z 244 (MH⁺).

52b (S)-2-Methylpiperazine-1-carboxamide

The title compound, obtained as a yellow oil (480 mg) was prepared fromthe product of Example 52a following the procedure of Example 7b. Thecrude product was carried forward without purification. R_(f)=0.1 (95:5dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); Mass spectrum(ESI+ve) m/z 144 (MH⁺).

52c(S,E)-2-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a clear oil (315 mg, 56%) was preparedfrom the product of Example 1 using the procedure of Example 3 exceptthe product of Example 52b was substituted for methylaminehydrochloride. [α]_(D)=16° (c=0.005, CHCl₃); R_(f)=0.43 (95:5dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H NMR (400MHz, CDCl₃) δ 7.41 (d, J=15.0 Hz, 1H), 6.29-6.10 (m, 1H), 4.57 (s, 3H),4.41-4.23 (m, 1H), 4.12-3.55 (m, 3H), 3.51 (d, J=5.0 Hz, 1H), 3.22 (s,3H), 2.94-2.79 (m, 1H), 2.06 (t, J=6.0 Hz, 2H), 1.76 (s, 3H), 1.71-1.59(m, 3H), 1.50 (m, 2H), 1.21 (s, 3H), 1.07 (s, 6H); Mass spectrum(ESI+ve) m/z 320 (MH⁺).

Example 53(R,E)-2-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piper-azine-1-carboxamide53a (R)-tert-Butyl 4-carbamoyl-3-methylpiperazine-1-carboxylate

The title compound, obtained as a yellow foam (377 mg, 65%) was preparedfrom (R)-1-Boc-3-methylpiperazine following the procedure of Example 7b.[α]_(D)=−35° (c=0.005, EtOH); R_(f)=0.48 (95:5dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H NMR (400MHz, CDCl₃) δ 4.50 (s, 2H), 4.23-3.52 (m, 5H), 3.10 (m, 2H), 3.01-2.78(m, 1H), 1.63 (s, 1H), 1.49 (s, 10H), 1.21 (d, J=6.0 Hz, 3H); Massspectrum (ESI+ve) m/z 244 (MH⁺).

53b (R)-2-Methylpiperazine-1-carboxamide

The title compound, obtained as a yellow oil (552 mg) was prepared fromthe product of Example 53a following the procedure of example 7b. Thecrude product was carried forward without purification. R_(f)=0.1 (95:5dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); Mass spectrum(ESI+ve) m/z 144 (MH⁺).

53c(R,E)-2-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a colorless amorphous solid (120 mg,31%) was prepared from the product of Example 1 using the procedure ofExample 3 except the product of Example 53b was substituted formethylamine hydrochloride. [α]_(D)=−11° (c=0.005, CHCl₃); R_(f)=0.33(93:7 dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H NMR(400 MHz, CDCl₃) δ 7.38 (d, J=15.5 Hz, 1H), 6.29-6.07 (m, 1H), 4.55 (s,2H), 4.38-4.21 (m, 1H), 3.94-3.09 (m, 6H), 2.04 (t, J=6.0 Hz, 2H), 1.74(s, 3H), 1.66-1.57 (m, 2H), 1.50-1.43 (m, 5H), 1.40 (d, J=6.0 Hz, 2H),1.18 (s, 3H), 1.05 (s, 6H); Mass spectrum (ESI+ve) m/z 320 (MH⁺).

Example 54(E)-N²-Methyl-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1,2-dicarboxamide54a4-((Benzyloxy)carbonyl)-1-(tert-butoxycarbonyl)piperazine-2-carboxylicacid

The tile compound was prepared according to the procedure of [Kempf, D.J.; Norbeck, D. W.; Sham, H. L. U.S. Pat. No. 5,455,351, Oct. 3, 1995].Piperazine-2-carboxylic acid (10.0 g, 77.0 mmol) was dissolved in a 1:1solution of 1,4-dioxane:water (100 mL) at room temperature with vigorousstirring. The clear solution was adjusted to pH 11 by the addition of anaqueous solution of sodium hydroxide (80 mL of a 1N solution). The pHwas monitored in situ with a pH meter throughout the reaction. Thereaction flask was fitted with an addition funnel that contained asolution of N-α-(benzyloxycarbonyloxy) succinamide (13.6 g, 55 mmol) in1,4-dioxane (50 mL). The N-α-(benzyloxycarbonyloxy) succinamide solutionwas added over 45 minutes at room temperature and the pH was kept above10 by the periodic addition of 1N sodium hydroxide. The pH of thesolution was adjusted to 9.5 and2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (13.4 g, 55 mmol)was added as a solution in 1,4-dioxane (50 mL) over 10 minutes. The pHwas maintained at 9.5 and the solution was stirred at room temperaturefor 17 hours. The solution was then acidified to pH 2 and the aqueoussolution was washed with diethyl ether (3×150 mL). The aqueous solutionwas cooled to 0° C. and acidified by adding of concentrated hydrochloricacid. The acidic solution was extracted with ethyl acetate (5×150 mL).The combined organic phases were dried over sodium sulfate, filtered andconcentrated in vacuo. The residue was triturated with a 1:1 solution ofdichloromethane: hexanes (150 mL) and the solvent was removed in vacuoto provide the product as a viscous yellow oil (15.7 g, 43 mmol, 80%).R_(f)=0.60 (66:34 dichloromethane:ethyl acetate+0.1% (v/v acetic acid);¹H-NMR (400 MHz, DMSO) δ 13.0 (br s, 1H), 7.37-7.36 (m, 5H), 5.05 (s,2H), 4.54-4.33 (m, 2H), 3.90-3.66 (m, 2H), 3.07-2.81 (m, 4H), 1.38 (s,9H); Mass spectrum (ESI+ve) m/z 365.1 (MH⁺).

54b 4-Benzyl 1-tert-butyl2-(methylcarbamoyl)piperazine-1,4-dicarboxylate

4-((Benzyloxy)carbonyl)-1-(tert-butoxycarbonyl)piperazine-2-carboxylicacid (1.70 g, 4.70 mmol), DMF (20 mL), diisopropylethylamine (2.50 mL,14.1 mmol) and methylamine hydrochloride (0.350 g, 5.20 mmol) were mixedtogether at room temperature under argon for 10 minutes.2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 2.00 g, 5.20 mmol) was then added to thereaction mixture in one portion. The mixture was stirred at roomtemperature under argon for 18 hours. The reaction mixture was thenpoured into water (100 mL) and extracted with ethyl acetate (4×25 mL).The combined organic phases were washed with saturated ammonium chloride(3×15 mL), water (3×15 mL) and brine (70 mL). The combined organicphases were then dried over sodium sulfate, filtered and concentrated invacuo. The product, obtained as a white foam (0.91 g, 2.4 mmol, 51%) waspurified by column chromatography (gradient elution 20:80 ethylacetate:hexanes to 100% ethyl acetate). R_(f)=0.10 (50:50 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.39-7.33 (m, 5H), 5.17 (brs, 2H), 4.68-4.58 (m, 2H), 3.98-3.88 (m, 2H), 3.23-3.08 (m, 4H), 2.07(s, 3H), 1.50 (s, 9H); Mass spectrum (ESI+ve) m/z 378.0 (MH⁺).

54c tert-Butyl 2-(methylcarbamoyl)piperazine-1-carboxylate

4-Benzyl 1-tert-butyl 2-(methylcarbamoyl)piperazine-1,4-dicarboxylate(0.910 g, 2.40 mmol) was dissolved in methanol (10 mL) at roomtemperature with stirring and the vial was flushed with argon. Palladiumon carbon (91.0 mg of 10 wt % on carbon) was added in one portion to thestirred reaction mixture. The reaction flask was charged with hydrogengas (1 atm) and stirred for 18 hours at room temperature. The palladiumon carbon was removed by vacuum filtration through Celite and rinsedwith additional methanol (5×10 mL). The combined filtrates wereconcentrated in vacuo. The product, obtained as a yellow solid (0.16 g,0.66 mmol, 27%) was purified by column chromatography (isocratic 3:97methanol:dichloromethane+0.1% (vv) ammonium hydroxide). R_(f)=0.32 (3:97methanol:dichloromethane+0.1% (v/v) ammonium hydroxide); ¹H-NMR (400MHz, CDCl₃) δ 6.41 (br s, 1H), 4.61-4.59 (m, 1H), 3.68-3.65 (m, 1H),3.18-2.85 (m, 6H), 2.47 (br s, 3H), 1.51 (s, 9H); Mass spectrum (ESI+ve)m/z 243.9 (MH⁺).

54d (E)-tert-Butyl2-(methylcarbamoyl)-4-(3-(2,6,6-trimethycyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate

(E)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylic acid (0.120 g, 0.620mmol), diisopropylethylamine (0.210 mL, 1.20 mmol), tert-butyl2-(methylcarbamoyl)piperazine-1-carboxylate (0.150 g, 0.620 mmol) weredissolved in a 1:5 mixture of dichloromethane: acetonitrile at roomtemperature under argon.2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 0.23 g, 0.62 mmol) was added in one portionand the reaction mixture was stirred at room temperature for 18 hours.The reaction mixture was passed through a carbonate SPE cartridge(silica-carbonate Silicycle, 2 g, 230-400 mesh) followed by filtrationthrough a tosic acid SPE cartridge (silica-tosic acid Silicycle, 1 g,230-400 mesh) and the solvent was removed in vacuo. The product,obtained as a clear oil (0.17 g, 0.41 mmol, 66%) was purified by columnchromatography (gradient elution 30:70 ethyl acetate:hexanes to 100%ethyl acetate). The proton NMR spectrum shows evidence that the productis a mixture of rotamers. R_(f)=0.14 (50:50 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.15-7.12 (m, 1H), 6.23-5.98 (m, 1H),4.60-4.27 (m, 1H), 4.23-4.10 (m, 2H), 3.98-3.87 (m, 2H), 3.82-3.62 (m,1H), 3.37-3.13 (m, 2H), 3.09-2.71 (m, 1H), 2.59 (s, 2H), 1.87 (s, 3H),1.61-1.58 (m, 2H), 1.45-1.42 (m, 2H), 1.30 (s, 9H), 0.88 (s, 6H); Massspectrum (ESI+ve) m/z 420.1 (MH⁺).

54e (E)-N²-methyl-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1,2-dicarboxamide

(E)-tert-Butyl2-(methylcarbamoyl)-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate(0.170 g, 0.410 mmol) was dissolved in dichloromethane (5 mL) at roomtemperature. Trifluoroacetic acid (3 mL) was added to the stirredsolution and the reaction mixture was stirred at room temperature for 1hour and then concentrated in vacuo to provide the crude trifluoroaceticacid salt as a viscous yellow oil (0.42 g). The trifluoroacetic acidsalt was analyzed by thin layer chromatography and LC-MS then useddirectly in the next reaction. R_(f)=0.17 (7:93methanol:dichloromethane+0.1% (v/v) ammonium hydroxide, ninhydrinstaining); Mass spectrum (ESI+ve) m/z 320.1 (MH⁺).

The trifluoroacetic acid salt (0.4 g) and potassium carbonate (0.54 g,3.9 mmol) were stirred at room temperature under argon for 20 minutes.Trimethylsilyl isocyanate (0.32 mL, 3.9 mmol) was added in one portionand the mixture was stirred for 18 hours at room temperature. Thereaction mixture was poured into saturated ammonium chloride (15 mL) andextracted with dichloromethane (3×10 mL). The combined organic phaseswere dried over sodium sulfate, filtered and concentrated in vacuo. Theproduct, obtained as a white solid (45 mg, 0.12 mmol, 32%) was purifiedby preparative thin layer chromatography (1000 μm thickness SiO₂ gel, 20cm×20 cm plate, eluent 10:90 methanol:dichloromethane+0.1% (v/v)ammonium hydroxide). The proton NMR spectrum shows evidence that theproduct is a mixture of rotamers. Mp=100-112° C.; R_(f)=0.62 (10:90methanol:dichloromethane+0.1% (v/v) ammonium hydroxide); ¹H-NMR (400MHz, CDCl₃) δ 7.43-7.34 (m, 1H), 6.47-6.40 (m, 1H), 5.18-4.61 (m, 4H),4.35-4.15 (m, 1H), 3.93-3.67 (m, 1H), 3.46-2.97 (m, 3H), 2.83-2.82 (m,3H), 2.10-2.06 (m, 2H), 1.80 (s, 3H), 1.65-1.61 (m, 2H), 1.51-1.48 (m,2H), 1.09 (m, 6H); Mass spectrum (ESI+ve) m/z 363.1 (MH⁺).

Example 55N¹-((2,6,6-Trimethylcyclohex-1-en-1-yl)methyl)piperazine-1,4-dicarboxamide55a tert-Butyl 4-(((2,6,6-Trimethylcyclohex-1-en-1-yl)methyl)carbamoyl)piperazine-1-carboxylate

To a stirred solution of 2-(2,6,6-trimethylcyclohex-1-en-1-yl)aceticacid (0.30 g, 1.6 mmol) in anhydrous benzene (16 mL) was addeddiphenylphosphoryl azide (0.45 g, 1.6 mmol) and triethylamine (0.51 g,4.8 mmol). The pale yellow reaction mixture was heated to reflux for 3hours until it turned a blue color. The reaction was cooled to roomtemperature and tert-butyl piperazine-1-carboxylate (0.31 g, 1.6 mmol)and triethylamine (0.17 g, 1.6 mmol) were added to the reaction mixture.The reaction was stirred at room temperature for 18 hours overnight.

The reaction mixture was diluted with ethyl acetate (150 mL),transferred to a separatory funnel and extracted with a 50% saturatedsolution of ammonium chloride (2×75 mL), and a saturated solution ofsodium bicarbonate (2×75 mL). the organic layer was then washed withbrine (75 mL), dried over magnesium sulfate, filtered and concentratedin vacuo to give a pale yellow solid (0.60 g, quantitative). R_(f)=0.65(25:75 ethyl acetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 4.00 (s, 1H),3.79 (d, J=4.0 Hz, 2H), 3.52-3.17 (m, 8H), 2.04-1.82 (m, 2H), 1.63 (s,3H), 1.60-1.54 (m, 2H), 1.44 (s, 11H), 0.98 (s, 6H); Mass spectrum(ESI+ve) m/z 366 (MH⁺).

55bN-((2,6,6-Trimethylcyclohex-1-en-1-yl)methyl)piperazine-1-carboxamide

The title compound, obtained as a yellow oil (265 mg, quantitative) wasprepared from the product of Example 55a by following the procedure ofexample 49b. The crude product was carried forward without purification.R_(f)=0.05 (95:5 chloroform:methanol); Mass spectrum (ESI+ve) m/z 266(MH⁺).

55cN¹-((2,6,6-Trimethylcyclohex-1-en-1-yl)methyl)piperazine-1,4-dicarboxamide

The title compound, obtained as a pale yellow solid (33 mg, 12%) wasprepared from the product of Example 55b following the procedure ofExample 33. Mp=168.9-169.7° C. R_(f)=0.45 (90:10 chloroform:methanol);¹H NMR (400 MHz, d₄-MeOD) δ 3.79-3.68 (m, 3H), 3.27 (s, 3H), 1.96 (t,J=6.0 Hz, 2H), 1.77-1.56 (m, 6H), 1.48-1.41 (m, 2H), 1.35-1.08 (m, 1H),0.99 (s, 6H), 0.93 (d, J=6.5 Hz, 1H), 0.88 (d, J=6.5 Hz, 1H), 0.06 (d,J=4.0 Hz, 1H); Mass spectrum (ESI+ve) m/z 308 (MH⁺).

Example 56 N¹-Methyl-N¹-((2,6,6-trimethylcyclohex-1-en-1-yl)methyl)piperazine-1,4-dicarboxamide 56aN-Methyl-N-((2,6,6-trimethylcyclohex-1-en-1-yl)methyl)-1H-imidazole-1-carboxamide

N-Methyl-1-(2,6,6-cyclohex-1-en-1-yl)methanamine hydrochloride (1.00 g,4.90 mmol) and triethylamine (0.750 mL, 5.40 mmol) were stirred underargon at room temperature in THF (12 mL) for 15 minutes.Carbonyldiimidazole (0.88 g, 5.4 mmol) was added in one portion to thestirred reaction mixture and the reaction mixture was heated to refluxfor 18 hours. The solvent was removed in vacuo and the residue wasdissolved in dichloromethane (100 mL). The organic phase was washed withwater (2×75 mL), dried over sodium sulfate, filtered and concentrated invacuo to afford the product as a yellow oil (1.2 g, 4.6 mmol, 93%).R_(f)=0.90 (7:93 methanol:dichloromethane+0.1% (v/v) ammoniumhydroxide); ¹H-NMR (400 MHz, CDCl₃) δ 7.90 (s, 1H), 7.24 (s, 1H), 7.10(s, 1H), 4.26 (s, 2H), 2.94 (s, 3H), 2.07-2.04 (m, 2H), 1.72 (s, 3H),1.68-1.62 (m, 2H), 1.50-1.47 (m, 2H), 1.03 (s, 6H); Mass spectrum(ESI+ve) m/z 261.9 (MH⁺).

56b3-Methyl-1-(methyl((2,6,6-trimethylcyclohex-1-en-1-yl))methyl)carbamoyl)-1H-imidazol-3-iumIodide

N-Methyl-N-((2,6,6-trimethylcyclohex-1-en-1-yl)methyl)-1H-imidazole-1-carboxamide(1.20 g, 4.60 mmol) and iodomethane (1.1 mL, 18 mmol) were dissolved inacetonitrile and stirred at room temperature for 18 hours. The reactionmixture was concentrated in vacuo to provide the product as ahygroscopic yellow foam (1.8 g, 4.4 mmol, 97%). ¹H-NMR (400 MHz, DMSO) δ9.63 (br s, 1H), 8.07 (br s, 1H), 7.87 (s, 1H), 4.24 (br s, 2H), 3.91(s, 3H), 2.88 (s, 3H), 2.10-2.08 (m, 2H), 1.70-1.59 (m, 5H), 1.46-1.44(m, 2H), 1.03-0.95 (m, 6H); Mass spectrum (ESI+ve) m/z 275.9 (MH⁺).

56cN¹-Methyl-N¹-((2,6,6-trimethylcyclohex-1-en-1-yl)methyl)piperazine-1,4-dicarboxamide

3-Methyl-1-(methyl((2,6,6-trimethylcyclohex-1-en-1-yl))methyl)carbamoyl)-1H-imidazol-3-iumiodide (0.200 g, 0.470 mmol), piperazine-1-carboxamide hydrochloride(78.0 mg, 0.470 mmol) and triethylamine (0.130 mL, 0.930 mmol) weredissolved in a 1:4 mixture of acetonitrile: dichloromethane. Thereaction mixture was stirred at room temperature under argon for 2 days.The reaction mixture was poured into saturated ammonium chloride (30 mL)and the organic layer was removed. The aqueous layer was extracted withdichloromethane (4×15 mL). The combined organic phases were dried oversodium sulfate, filtered and concentrated in vacuo. The product obtainedas a white solid (11 mg, 0.03 mmol, 7%) was purified by preparativeplate thin layer chromatography (1000 μm thickness SiO₂ gel, 20 cm×20 cmplate, eluent 10:90 methanol:ethyl acetate+0.1% (v/v) ammoniumhydroxide. Mp=139.6-140.3° C.; R_(f)=0.62 (10:90 methanol:ethylacetate+0.1% (v/v) ammonium hydroxide); ¹H-NMR (400 MHz, DMSO) δ 6.00(s, 2H), 3.93 (s, 2H), 3.32-3.28 (m, 4H), 3.01-3.00 (m, 4H), 2.66 (s,3H), 1.99-1.96 (m, 2H), 1.65 (s, 3H), 1.59-1.57 (m, 2H), 1.41-1.40 (m,2H), 0.96 (s, 6H); Mass spectrum (ESI+ve) m/z 323.0 (MH⁺).

Example 57 (R,E)-1-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)pyrrolidin-3-yl carbamate 57a(R,E)-1-(3-Hydroxypyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one

The title compound, obtained as a colorless oil (0.35 g, 86%), wasprepared by following the procedure of Example 3, except(R)-pyrrolidin-3-ol was substituted for methylamine hydrochloride.[α]_(D) ²³=−13.78° (c=0.005, methanol); R_(f)=0.2 (100% ethyl acetate);¹H-NMR (400 MHz, CDCl₃) δ 7.36 (d, J=15.5 Hz, 1H), 6.09 (d, J=15.5 Hz,1H), 4.55 (m, 1H), 3.80-3.53 (m, 4H), 2.95 (m, 1H), 2.12-1.97 (m, 4H),1.76 (s, 3H), 1.63 (m, 2H), 1.48 (m, 2H), 1.07 (s, 6H); Mass spectrum(ESI+ve) m/z 264 (MH⁺).

57b (R,E)-1-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)pyrrolidin-3-yl carbamate

Trichloroacetyl isocyanate (501 mg, 2.66 mmol) was added to a solutionof 57a (350 mg, 1.33 mmol) in tetrahydrofuran (3 mL). The solution wasstirred for 12 hours at room temperature and then treated with water(0.5 mL) to destroy the excess of trichloroacetyl isocyanate. Thereaction mixture was diluted with ethyl acetate (50 mL) and extractedwith water (30 mL). The organic layer was separated, dried overmagnesium sulfate, filtered and concentrated in vacuo. The crude productwas carried forward without further purification.

A solution of potassium carbonate (367 mg, 2.66 mmol) in water (8 mL)was added to a solution of the crude material (600 mg, 1.33 mmol) interahydrofuran (10 mL) and methanol (10 mL). The mixture was stirred for3 hours at room temperature, and then diluted with ethyl acetate (60 mL)and water (60 mL). The organic layer was separated, dried over magnesiumsulfate, filtered and concentrated in vacuo to yield the title compoundas an off-white solid (72 mg, 18%); [α]_(D) ²³=−12.42° (c=0.006,chloroform); R_(f)=0.2 (10:90 methanol:chloroform); ¹H-NMR (400 MHz,CDCl₃) δ 7.41 (d, J=15.5 Hz, 1H), 6.08 (d, J=15.5 Hz, 1H), 5.31 (m, 1H),4.72 (m, 2H), 3.88-3.77 (m, 2H), 3.77-3.62 (m, 2H), 2.18 (m, 2H), 2.05(m, 2H), 1.77 (s, 3H), 1.64 (m, 2H), 1.49 (m, 2H), 1.08 (m, 6H); Massspectrum (ESI+ve) m/z 307 (MH⁺).

Example 58 (S,E)-1-(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)pyrrolidin-3-yl)urea 58a (S,E)-tert-Butyl(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl) pyrrolidin-3-yl)carbamate

The title compound, obtained as a colorless oil (62 mg, 35%), wasprepared by following the procedure of Example 3, except (S)-tert-butylpyrrolidin-3-yl carbamate was substituted for methylamine hydrochloride.R_(f)=0.15 (100% ethyl acetate); ¹H-NMR (400 MHz, CDCl₃) δ 7.39 (dd,J=15.5, 6.0 Hz, 1H), 6.14-6.00 (m, 1H), 4.28 (m, 1H), 3.81 (m, 2H),3.73-3.32 (m, 4H), 2.32-2.12 (m, 1H), 2.06 (s, 3H), 1.83 (d, J=6.5 Hz,1H), 1.79 (s, 3H), 1.65 (m, 4H), 1.48 (m, 9H), 1.07 (m, 6H); Massspectrum (ESI+ve) m/z 363 (MH⁺).

58b(S,E)-1-(3-Aminopyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one

The title compound, obtained as white solid (42 mg, 98%), was preparedfrom the product of Example 58a by following the procedure of Example49b. R_(f)=0.05 (10:89:1 methanol:dichloromethane:ammonium hydroxide);¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (br s, 3H), 7.15 (d, J=16.0 Hz, 1H),6.24-6.09 (m, 1H), 3.95-3.51 (m, 4H), 2.33-2.19 (m, 1H), 2.04 (m, 4H),1.73 (s, 3H), 1.65-1.53 (m, 2H), 1.51-1.38 (m, 2H), 1.03 (s, 3H), 1.01(s, 3H); Mass spectrum (ESI+ve) m/z 263 (MH⁺).

58c(S,E)-1-(1-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)pyrrolidin-3-yl)urea

The title compound, obtained as white film (6.7 mg, 12%), was preparedfrom the product of Example 58b by following the procedure of Example57b. R_(f)=0.2 (5:95 methanol:chloroform); ¹H NMR (400 MHz,) δ 7.13 (d,J=15.5 Hz, 1H), 6.31 (m, 1H), 6.14 (d, J=15.5 Hz, 1H), 5.45 (m, 2H),4.08 (m, 1H), 3.58 (t, J=7.0 Hz, 1H), 3.53-3.14 (m, 4H), 2.10-1.95 (m,2H), 1.73 (s, 3H), 1.58 (m, 2H), 1.46 (m, 2H), 1.03 (m, 6H); Massspectrum (ESI+ve) m/z 306 (MH⁺).

Example 59 (R,E)-1-(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)pyrrolidin-3-yl)urea 59a (R,E)-tert-Butyl(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl) pyrrolidin-3-yl)carbamate

The title compound, obtained as a colorless oil (300 mg, 65%), wasprepared by following the procedure of Example 3, except (R)-tert-butylpyrrolidin-3-ylcarbamate was substituted for methylamine hydrochloride.R_(f)=0.15 in (40:60 ethyl acetate:hexane); ¹H-NMR (400 MHz, CDCl₃) δ7.39 (dd, J=15.5, 6.0 Hz, 1H), 6.14-6.00 (m, 1H), 4.28 (m, 1H), 3.81 (m,2H), 3.73-3.32 (m, 4H), 2.32-2.12 (m, 1H), 2.06 (s, 3H), 1.83 (d, J=6.5Hz, 1H), 1.79 (s, 3H), 1.65 (m, 4H), 1.48 (m, 9H), 1.07 (m, 6H); Massspectrum (ESI+ve) m/z 363 (MH⁺).

59b(R,E)-1-(3-Aminopyrrolidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one

The title compound, obtained as white solid (70 mg, 84%), was preparedfrom the product of Example 59b by following the procedure of Example49b. R_(f)=0.05 (10:89:1 methanol:dichloromethane:ammonium hydroxide);¹H NMR (400 MHz, DMSO-d₆) δ 8.4 (br s, 3H), 7.15 (d, J=16.0 Hz, 1H),6.24-6.09 (m, 1H), 3.95-3.51 (m, 4H), 2.33-2.19 (m, 1H), 2.04 (m, 4H),1.73 (s, 3H), 1.65-1.53 (m, 2H), 1.51-1.38 (m, 2H), 1.01 (m, 6H); Massspectrum (ESI+ve) m/z 263 (MH⁺).

59c (R,E)-1-(1-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)pyrrolidin-3-yl)urea

The title compound, obtained as white film (21 mg, 10%), was preparedfrom the product of Example 59b by following the procedure of Example57b: R_(f)=0.2 (5:95 methanol:chloroform); ¹H NMR (400 MHz, DMSO-d₆) δ7.13 (d, J=15.5 Hz, 1H), 6.31 (m, 1H), 6.14 (d, J=15.5 Hz, 1H), 5.45 (m,2H), 4.08 (m, 1H), 3.58 (t, J=7.0 Hz, 1H), 3.53-3.14 (m, 4H), 2.10-1.95(m, 2H), 1.73 (s, 3H), 1.58 (m, 2H), 1.46 (m, 2H), 1.03 (m, 6H); Massspectrum (ESI+ve) m/z 306 (MH⁺).

Example 60(E)-4-(3-(2,6,6-Trimethyl-3-oxocyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide60a (E)-Methyl 3-(2,6,6-trimethylcyclohex-1-enyl)acrylate

(E)-3-(2,6,6-Trimethylcyclohex-1-enyl)acrylic acid (5.10 g, 26.3 mmol)was dissolved in acetone (20 mL) and anhydrous potassium carbonate (3.66g, 26.5 mmol) was added and the reaction mixture was stirred vigorously.Methyl iodide (4.11 g, 1.80 mL, 28.9 mmol) was added via syringe and thereaction mixture was stirred at room temperature for 3 days.

The reaction was dissolved in diluted with diethyl ether (175 mL) thenextracted with distilled water (100 mL), saturated sodium bicarbonate(100 mL) and brine (100 mL). The combined aqueous layers were extractedwith diethyl ether (2×75 mL) and the combined organic layers were driedover sodium sulfate and concentrated in vacuo to yield a viscous yellowoil (5.17 g). The product was purified by flash chromatography to yielda clear oil (3.77 g, 70%). R_(f)=0.21 (20:80 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.42 (d, J=16.0 Hz, 1H), 5.80 (d, J=16.0 Hz,1H), 3.74 (s, 3H), 2.03 (t, J=6.4 Hz, 2H), 1.72 (s, 3H), 1.61-1.58 (m,2H), 1.47-1.44 (m, 2H), 1.04 (s, 6H); Mass spectrum (ESI+ve) m/z 209(MH⁺).

60b (E)-Methyl 3-(2,6,6-trimethyl-3-oxocyclohex-1-enyl)acrylate

(E)-Methyl 3-(2,6,6-trimethylcyclohex-1-enyl)acrylate (3.77 g, 18.0mmol) was dissolved in 1,4-dioxane (60 mL) to which selenium dioxide(2.00 g, 18.0 mmol) was added and allowed to stir vigorously. Thereaction mixture was sealed with a rubber septum and placed into an 80°C. oil bath for 16 hours.

The reaction was filtered and concentrated in vacuo to yield a brown oil(5.20 g). The product was purified by flash column chromatography toyield a yellow oil (440 mg, 11%). R_(f)=0.3 (10:90 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.41 (d, J=16.4 Hz, 1H),5.92 (d, J=16.4 Hz, 1H), 3.80 (s, 3H), 2.51 (d, J=6.8 Hz, 2H), 1.88 (d,J=7.2 Hz, 2H), 1.80 (s, 3H), 1.18 (s, 6H); Mass spectrum (ESI+ve) m/z223 (MH⁺).

60c (E)-3-(2,6,6-Trimethyl-3-oxocyclohex-1-enyl)acrylic acid

(E)-Methyl 3-(2,6,6-trimethyl-3-oxocyclohex-1-enyl)acrylate (830 mg,4.50 mmol) was dissolved in tetrahydrofuran (30 mL) at room temperatureunder argon to which a solution of lithium hydroxide (210 mg, 5.00 mmol)in water (5 mL) was added. The reaction was stirred vigorously at roomtemperature under argon for 2 hours.

The reaction was acidified with 1M hydrochloric acid solution at 0° C.,diluted with water (150 mL) and extracted with ethyl acetate (3×85 mL).The combined organic layers were washed with brine (120 mL) and driedover sodium sulfate. The solvent concentrated in vacuo to yield a yellowoil (427 mg, 55%). ¹H-NMR (400 MHz, CDCl₃) δ 7.49 (d, J=16.0 Hz, 1H),5.95 (d, J=16.4 Hz, 1H), 2.54 (t, J=6.8 Hz, 2H), 1.89 (t, J=6.8 Hz, 2H),1.81 (s, 3H), 1.19 (s, 6H); Mass spectrum (ESI+ve) m/z 209 (MH⁺).

60d (E)-tert-Butyl4-(3-(2,6,6-trimethyl-3-oxocyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a clear oil (60.0 mg, 26%), was preparedfrom the product of Example 60c by following the procedure of Example 3.R_(f)=0.25 (50:50 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.38(d, J=15.6, 1H), 6.32 (d, J=15.6 Hz, 1H), 3.97-3.48 (m, 8H), 2.52 (t,J=6.8 Hz, 2H), 1.88 (t, J=6.8 Hz, 2H), 1.81 (s, 3H), 1.41 (s, 9H), 1.18(s, 6H); Mass spectrum (ESI+ve) m/z 377 (MH⁺).

60e(E)-4-(3-(2,6,6-Trimethyl-3-oxocyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide

The product of example 60d dissolved in dichloromethane and a 4.0 Msolution of hydrochloric acid in 1,4-dioxane was added to the stirredreaction mixture. The reaction was stirred at room temperature for 4hours and then concentrated in vacuo to afford a pale yellow crude oil.The title compound, obtained as a white film (23.0 mg, 45%), wasprepared from the product of Example 60d by following the procedure ofExample 33. R_(f)=0.50 (10:90 methanol:chloroform); ¹H-NMR (400 MHz,CDCl₃) δ 7.39 (d, J=15.6 Hz, 1H), 6.32 (d, J=15.6 Hz, 1H), 4.64 (s, 2H),3.76-3.45 (m, 8H), 2.52 (t, J=6.8 Hz, 2H), 1.88 (t, J=6.8 Hz, 2H), 1.80(s, 3H), 1.18 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 319.9 (MH⁺).

Example 61 (E)-4-(3-(3-Hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide 61a(E)-2,4,4-Trimethyl-3-(3-oxobut-1-en-1-yl)cyclohex-2-en-1-yl acetate

To a solution of 1,4-benzoquinone (6.00 g, 55.5 mmol) and beta-ionone(10.6 g, 55.5 mmol) in acetic acid (180 mL) were added palladiumbis(trifluoroacetate) (900 mg, 3.00 mmol) and o-methoxyacetophenone(1.68 g, 11.1 mmol). The mixture was heated to 70° C. for 12 hours. Thesolvent was concentrated in vacuo and then a solution of sodiumhydroxide (200 mL, 6 N) was added, and the aqueous phase extracted withdiethyl ether (5×50 mL). The combined organic extracts were washed witha saturated solution of sodium carbonate (100 mL), dried over sodiumsulfate, filtered and concentrated in vacuo. The crude product waspurified by flash column chromatography (90:10 hexane:diethyl ether) toyield the title compound as a brown oil (7.8 g; 56%). R=0.25 (30:70diethyl ether:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.20 (d, J=16.5 Hz,1H), 6.15 (d, J=16.5 Hz, 1H), 5.25 (m, 1H), 2.33 (s, 3H), 2.10 (s, 3H),2.00-1.88 (m, 1H), 1.81-1.73 (m, 1H), 1.72 (s, 3H), 1.71-1.60 (m, 1H),1.48 (m, 1H), 1.09 (s, 3H), 1.04 (s, 3H); Mass spectrum (ESI+ve) m/z 251(MH⁺).

61b (E)-3-(3-Hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)acrylic acid

The title compound, obtained as clear yellow oil, was prepared from theproduct of Example 61a by following the procedure of Example 1, except(E)-2,4,4-trimethyl-3-(3-oxobut-1-en-1-yl)cyclohex-2-en-1-yl acetate wassubstituted for beta-ionone. ¹H NMR (400 MHz, CDCl₃) δ 7.51-7.45 (m,1H), 5.89 (d, J=16.0 Hz, 1H), 4.06 (t, J=4.5 Hz, 1H), 1.99-1.90 (m, 2H),1.90 (s, 3H), 1.80-1.63 (m, 2H), 1.47 (m, 1H), 1.10 (s, 3H), 1.07 (s,3H).

61c (E)-tert-Butyl4-(3-(3-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a colorless oil (50 mg, 98%), wasprepared by following the procedure of Example 3, except tert-butylpiperazine-1-carboxylate was substituted for methylamine hydrochlorideand (E)-3-(3-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)acrylic acid wassubstituted for (E)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylic acid.R_(f)=0.10 (50:50 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.33(s, 1H), 6.25 (d, J=15.5 Hz, 1H), 4.03 (t, J=4.5 Hz, 1H), 3.66 (m, 2H),3.62-3.51 (m, 2H), 3.49 (m, 4H), 2.00-1.89 (m, 1H), 1.88 (s, 3H),1.78-1.63 (m, 4H), 1.53-1.42 (m, 9H), 1.07 (s, 6H); Mass spectrum(ESI+ve) m/z 379 (MH⁺).

61d (E)-4-(3-(3-Hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl) acryloyl)piperazine-1-carboxamide

The product of example 61c dissolved in dichloromethane and a 4.0 Msolution of hydrochloric acid in 1,4-dioxane was added to the stirredreaction mixture. The reaction was stirred at room temperature for 4hours and then concentrated in vacuo to afford a pale yellow crude oil.The title compound, obtained as colorless oil (8 mg, 4%), was preparedfrom the product of Example 61c by following the procedure of Example33. R_(f)=0.15 (5:95 methanol:chloroform); ¹H NMR (400 MHz, DMSO-d₆) δ7.34 (d, J=15.5 Hz, 1H), 6.25 (d, J=15.5 Hz, 1H), 5.81-5.53 (m, 2H),4.05 (m, 1H), 3.81 (m, 2H), 3.65 (m, 2H), 3.54 (m, 4H), 1.98 (m, 1H),1.87 (s, 3H), 1.80-1.65 (m, 2H), 1.53-1.43 (m, 1H), 1.07 (s, 6H); Massspectrum (ESI+ve) m/z 322 (MH⁺).

Example 62(E)-4-(3-(3,3-Difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide62a (E)-Methyl3-(6,8,8-trimethyl-1,4-dithiaspiro[4.5]dec-6-en-7-yl)acrylate

(E)-Methyl 3-(2,6,6-trimethyl-3-oxocyclohex-1-en-1-yl)acrylate (0.550 g,2.40 mmol) was dissolved in 1,2-ethanedithiol (0.710 mL, 8.50 mmol) atroom temperature under argon. The homogenous mixture was then cooled to−15° C. and stirred for 10 minutes. Zinc(II)chloride (17.0 mg, 0.120mmol) was added in one portion and the mixture was stirred at −15° C.for 3 hours and then at room temperature for 18 hours. The reactionmixture was then diluted with water (30 mL). The aqueous layer wasextracted with ethyl acetate (3×20 mL). The combined organic phases weredried over sodium sulfate, filtered and concentrated in vacuo. Theproduct, obtained as white crystals (0.6 g, 2.0 mmol, 85%) was purifiedby column chromatography (isocratic 5% ethyl acetate:hexanes).Mp=79.5-88.1° C.; R_(f)=0.70 in (15:85 ethyl acetate:hexanes); ¹H-NMR(400 MHz, CDCl₃) δ 7.36 (d, J=16.2 Hz, 1H), 5.87 (d, J=16.2 Hz, 1H),3.79 (s, 3H), 3.42-3.31 (m, 4H), 2.32-2.25 (m, 2H), 2.00 (s, 3H),1.75-1.69 (m, 2H), 1.58 (s, 2H), 1.06 (s, 6H); Mass spectrum (ESI+ve)m/z 298.8.

62b (E)-Methyl3-(3,3-difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acrylate

A slurry of N-iodosuccinamide (1.12 g, 5.00 mmol) and dichloromethane (6mL) in a Nalgene bottle was cooled to −78° C. under argon. Hydrofluoricacid pyridine complex (1.40 mL, 49.6 mmol) was slowly added to theslurry and stirred for 10 minutes under argon. A solution of (E)-Methyl3-(6,8,8-trimethyl-1,4-dithiaspiro[4.5]dec-6-en-7-yl)acrylate (62a,0.370 g, 1.20 mmol) in dichloromethane (1 mL) was added to the reactionmixture and stirred for 1 hour at −78° C. The reaction mixture waspoured into a 1:1 solution of saturated sodium sulfite: saturated sodiumbicarbonate (100 mL). The aqueous solution was extracted with ethylacetate (3×70 mL). The combined organic extracts were dried over sodiumsulfate, filtered and concentrated in vacuo. The product, obtained as abright yellow oil (0.16 g, 0.65 mmol, 51%) was purified by columnchromatography (isocratic 10% ethyl acetate: hexanes). R_(f)=0.71 in(10:60 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.52 (d, J=16.2Hz, 1H), 5.90 (d, J=16.2 Hz), 3.80 (s, 3H), 2.18-2.12 (m, 2H), 1.80 (s,3H), 1.70-1.68 (m, 2H), 1.09 (s, 6H); ¹⁹F-NMR (376 MHz, CDCl₃) δ −93.6(ddd, J=3.0, 14.0, 14.0 Hz).

62c (E)-3-(3,3-difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acrylic acid

(E)-Methyl 3-(3,3-difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acrylate(62b, 57.0 mg, 0.230 mmol) was dissolved in a 3:1 solution oftetrahydrofuran: water at room temperature under argon. A solution oflithium hydroxide (0.25 mL, 0.25 mmol, 1M aqueous solution) was added atroom temperature and the reaction was stirred for 6 hours. The reactionmixture was poured into saturated ammonium chloride (20 mL) andextracted with dichloromethane (3×15 mL). The combined organic extractswere dried over sodium sulfate, filtered and concentrated in vacuo. Thisprovided the product as white crystals (54 mg, quantitative).Mp=114.3-119.5° C.; R_(f)=0.12 in (10:90 ethyl acetate:hexanes); ¹H-NMR(400 MHz, CDCl₃) δ 7.39 (d, J=16 Hz, 1H), 5.91 (d, J=16 Hz, 1H),2.20-2.09 (m, 2H), 1.81 (s, 3H), 1.70-1.67 (m, 2H), 1.09 (s, 6H);¹⁹F-NMR (376 MHz, CDCl₃) δ −94.1 (t, J=14.0 Hz).

62d (E)-(9H-fluoren-9-yl)methyl4-(3-(3,3-difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a clear oil (0.11 g, 81%), was preparedfrom the product of Example 62c by following the procedure of Example 3except (9H-fluoren-9-yl)methyl piperazine-1-carboxylate hydrochloridewas substituted for methylamine hydrochloride. R_(f)=0.35 in (30:70ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.80 (d, J=7.6 Hz,2H), 7.58 (d, J=7.2 Hz, 2H), 7.44 (t, J=7.6 Hz, 2H), 7.35 (t, J=7.6 Hz,2H), 7.26 (d, J=16 Hz, 1H), 6.28 (d, J=16 Hz, 1H), 4.54 (d, J=6 Hz, 2H),4.27 (t, J=6 Hz, 1H), 3.64-3.40 (m, 8H), 2.22-2.12 (m, 2H), 1.82 (s,3H), 1.72-1.69 (m, 2H), 1.10 (s, 6H); ¹⁹F-NMR (376 MHz, CDCl₃) δ −93.8(m); Mass spectrum (ESI+ve) m/z 521.1 (MH⁺).

62e(E)-3-(3,3-difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)-1-(piperazin-1-yl)prop-2-en-1-one

(E)-(9H-fluoren-9-yl)methyl 4-(3-(3,3-difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate (97.0 mg, 0.190 mmol), piperidine (0.4 mL, 20%v/v) and acetonitrile (2 mL) were stirred together at room temperaturefor 5 minutes. The reaction mixture was concentrated in vacuo and theresidue was lyophilized for 18 hours. The product, obtained as a clearoil (37 mg, 0.12 mmol, 67%) was purified by column chromatography(isocratic elution 5:95 methanol:dichloromethane+0.1% (v/v) ammoniumhydroxide. R_(f)=0.36 in (5:95 methanol:dichloromethane+0.1% (v/v)ammonium hydroxide; ¹H-NMR (400 MHz, CDCl₃) δ 7.20 (d, J=16 Hz, 1H),6.28 (d, J=16 Hz), 3.71-3.68 (m, 2H), 3.59-3.49 (m, 2H), 2.95-2.86 (m,4H), 2.19-2.09 (m, 2H), 1.80 (s, 3H), 1.69-1.66 (m, 2H), 1.07 s, 6H);¹⁹F-NMR (376 MHz, CDCl₃) δ −93.3 (ddd, J=3.3, 14.0, 14.0 Hz); Massspectrum (ESI+ve) m/z 299.1 (MH⁺).

62f: (E)-4-(3-(3,3-Difluoro-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as white crystals (21 mg, 68%), wasprepared from the product of Example 62e by following the procedure ofExample 33. Mp=181.3-182.4° C.; R_(f)=0.35 in (5:95methanol:dichloromethane+0.1% ammonium hydroxide; ¹H-NMR (400 MHz,CDCl₃) δ 7.26 (d, J=16 Hz, 1H), 6.29 (d, J=16 Hz, 1H), 3.80-3.40 (m,8H), 2.24-2.10 (m, 2H), 1.95-1.86 (m, 2H), 1.81 (br s, 3H), 1.74-1.66(m, 2H), 1.09 (s, 6H); ¹⁹F-NMR (376 MHz, CDCl₃) δ −93.8 (t, J=14.0 Hz);Mass spectrum (ESI+ve) m/z 342.0 (MH⁺).

Example 63(E)-4-(3-(3,3-Dideutero-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide 63a (E)-Methyl3-(3-deutero-3-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)acrylate

To a stirred solution of (E)-methyl3-(2,6,6-trimethyl-3-oxocyclohex-1-en-1-yl) acrylate (60b, 0.27 g, 1.2mmol) in d₄-methanol (6 mL) at 0° C. was added sodium borodeuteride(0.051 g, 1.2 mmol) in one portion. The reaction was stirred at 0° C.for 3 hours then quenched by adding a 50% saturated solution of ammoniumchloride (25 mL) and ethyl acetate (50 mL). The biphasic reactionmixture was transferred to a separatory funnel and separated, and theorganic layer was further washed with water (25 mL) and brine (25 mL),and then dried over magnesium sulfate, filtered and concentrated invacuo to give a pale yellow oil (0.24 g, 88%). R^(f)=0.33 (25:75 ethylacetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.34 (d, J=16.5 Hz, 1H),5.84 (d, J=16.5 Hz, 1H), 3.76 (s, 3H), 1.90 (ddd, J=13.5, 11.0, 3.0 Hz,1H), 1.84 (s, 3H), 1.72 (ddd, J=13.5, 7.0, 2.0 Hz, 1H), 1.64 (m, 1H),1.44 (ddd, J=13.5, 7.0, 3.0 Hz, 1H), 1.23 (br s, 1H), 1.05 (s, 3H), 1.03(s, 3H); Mass spectrum (ESI+ve) m/z 226 (MH⁺).

63b (E)-Methyl3-(3-acetoxy-3-deutero-2,6,6-trimethylcyclohex-1-en-1-yl)acrylate

To a solution of (E)-methyl3-(3-deutero-3-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)acrylate (63a,0.22 g, 0.98 mmol) in acetic anhydride (3 mL) was addedN,N-dimethylaminopyridine (0.012 g, 0.098 mmol). The reaction wasstirred at room temperature for 18 hours overnight. The reaction wasdiluted with ethyl acetate (25 mL) and transferred to a separatoryfunnel. The organic layer was extracted with a 1M solution of sodiumhydroxide (2×25 mL), washed with water (25 mL) and brine (25 mL), andthen dried over magnesium sulfate, filtered and concentrated in vacuo togive a pale yellow oil (0.20 g, 78%). R_(f)=0.45 (25:75 ethylacetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.34 (d, J=16.0 Hz, 1H),5.85 (d, J=16.0 Hz, 1H), 3.77 (s, 3H), 2.07 (s, 3H), 1.90 (ddd, J=13.5,11.0, 3.0 Hz, 1H), 1.73 (ddd, J=10.0, 7.0, 3.5 Hz, 1H), 1.69 (s, 3H),1.66-1.58 (m, 1H), 1.44 (ddd, J=13.5, 7.0, 3.0 Hz, 1H), 1.07 (s, 3H),1.03 (s, 3H); Mass spectrum (ESI+ve) m/z 208 (M-OAc⁺).

63c (E)-Methyl3-(3,3-dideutero-2,6,6-trimethylcyclohex-1-en-1-yl)acrylate

To a solution of (E)-methyl3-(3-acetoxy-3-deutero-2,6,6-trimethylcyclohex-1-en-1-yl)acrylate (0.19g, 0.71 mmol) in anhydrous tetrahydrofuran (18 mL) was added palladiumtetrakistriphenylphosphine (0.55 g, 0.48 mmol) and sodium borodeuteride(0.13 g, 3.1 mmol). The reaction flask was sealed tightly to allow buildup of pressure from the liberated deuterium gas, and the reaction wasstirred at room temperature for 18 hours overnight. The reaction wasquenched by adding a 50% saturated solution of ammonium chloride (20 mL)and diethyl ether (50 mL). The biphasic reaction mixture was filteredthrough Celite into a separatory funnel and separated, and the organiclayer was further washed with a 50% saturated solution of ammoniumchloride (50 mL) and brine (50 mL), then dried over magnesium sulfate,filtered and concentrated in vacuo to give a pale yellow oil (0.10 g,68%) which was carried forward without purification. R_(f)=0.90 (25:75ethyl acetate:hexanes).

63d (E)-3-(3,3-Dideutero-2,6,6-trimethylcyclohex-1-en-1-yl)acrylic acid

The title compound, obtained as a pale yellow solid (68 mg, 92%) wasprepared from the product of Example 63c following the procedure ofExample 60c. The compound was carried forward without purification.R_(f)=0.45 (25:75 ethyl acetate:hexanes).

63e (E)-tert-Butyl4-(3-(3,3-dideutero-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a pale yellow solid (126 mg,quantitative) was prepared from the product of Example 63d following theprocedure of Example 3 except tert-butyl piperazine-1-carboxylate wassubstituted for methylamine hydrochloride. R_(f)=0.55 (25:75 ethylacetate:hexanes); Mass spectrum (ESI+ve) m/z 365 (MH⁺).

63f(E)-3-(3,3-dideutero-2,6,6-trimethylcyclohex-1-en-1-yl)-1-(piperazin-1-yl)prop-2-en-1-one

The title compound, obtained as a yellow oil (51 mg, 56%) was preparedfrom the product of Example 63e by following the procedure of example49b. The crude product was carried forward without purification.R_(f)=0.30 (90:10 chloroform:methanol); Mass spectrum (ESI+ve) m/z 266(MH⁺).

63g (E)-4-(3-(3,3-dideutero-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as an off-white solid (35 mg, 59%) wasprepared from the product of Example 63f following the procedure ofExample 33 except potassium carbonate was substituted for triethylamine.Mp=148.4-149.7° C.; R_(f)=0.50 (90:10 chloroform:methanol); ¹H NMR (400MHz, CDCl₃) δ 7.34 (d, J=15.5 Hz, 1H), 6.17 (d, J=15.5 Hz, 1H), 4.88 (s,2H), 3.79-3.35 (m, 8H), 1.72 (s, 3H), 1.62-1.53 (m, 2H), 1.45 (dd,J=7.5, 4.0 Hz, 2H), 1.03 (s, 6H); Mass spectrum (ESI+ve) m/z 308 (MH⁺).

Example 64(E)-N-(piperidin-4-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamidehydrochloride 64a (E)-tert-Butyl4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamido)piperidine-1-carboxylate

The title compound, obtained as a clear oil (202 mg, 52%), was preparedfrom the product of Example 1a by following the procedure of Example 3except tert-butyl 4-aminopiperidine-1-carboxylate was substituted formethylamine hydrochloride. R_(f)=0.20 (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.30 (d, J=15.2, 1H), 5.70 (d, J=15.2 Hz, 1H),5.42-5.28 (m, 1H), 4.06-4.01 (m, 3H), 2.90-2.84 (m, 2H), 2.03-1.94 (m,4H), 1.72 (s, 3H), 1.59 (t, J=5.6 Hz, 2H), 1.58-1.47 (m, 11H), 1.34-1.31(m, 2H), 0.99 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 376.9 (MH⁺).

64b(E)-N-(Piperidin-4-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamidehydrochloride

The title compound, obtained as a yellow solid (166 mg, 99%), wasprepared by following the procedure of Example 49b. R_(f)=0.1 (90:10dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H-NMR (400MHz, CDCl₃) δ7.31 (d, J=16.0, 1H), 5.99 (d, J=16.0 Hz, 1H), 4.06-4.03(m, 1H), 3.65 (s, 2H), 3.46-3.43 (m, 2H), 3.34-3.30 (m, 2H), 2.22-2.01(m, 4H), 1.81-1.77 (m, 5H) 1.64-1.63 (m, 2H), 1.43-1.41 (m, 2H), 1.07(s, 6H); Mass spectrum (ESI+ve) m/z 277 (MH⁺).

Example 65(E)-N-Methyl-N-(piperidin-3-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamidehydrochloride 65a (E)-tert-Butyl3-(N-methyl-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamido)piperidine-1-carboxylate

The title compound, obtained as a yellow solid (335 mg, 83%), wasprepared from the product of Example 1a by following the procedure ofExample 3 except tert-butyl 3-(methylamino)piperidine-1-carboxylate wassubstituted for methylamine hydrochloride. R_(f)=0.20 (25:75 ethylacetate: hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.33 (d, J=15.0, 1H), 6.50(d, J=15.0 Hz, 1H), 4.44-3.78 (m, 4H), 2.95 (s, 3H), 2.84-2.78 (m, 1H),2.58-2.56 (m, 1H), 2.04-2.02 (m, 2H), 1.75 (s, 4H), 1.63-1.60 (m, 4H),1.48-1.44 (m, 11H), 1.01 (s, 6H); Mass spectrum (ESI+ve) m/z 391 (MH⁺).

65b(E)-N-Methyl-N-(piperidin-3-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamide hydrochloride

The title compound, obtained as a yellow oil (278 mg, 99%), was preparedfollowing the procedure of Example 49b. R_(f)=0.2 (90:10dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H-NMR (400MHz, CDCl₃) δ 7.31 (d, J=15.0, 1H), 6.34 (d, J=15.0 Hz, 1H), 4.81-4.76(m, 1H), 3.39-3.31 (m, 4H), 3.07-2.78 (m, 4H), 2.09 (t, J=6.0 Hz, 3H),2.05-1.82 (m, 3H), 1.79 (s, 3H), 1.53-1.49 (m, 2H), 1.28-1.24 (m, 2H),1.08 (s, 6H); Mass spectrum (ESI+ve) m/z 291 (MH⁺).

Example 66(E)-N-(Piperidin-3-yl)-3-(2,6,6-trimethylcyclohex-1-on-1-yl)acrylamidehydrochloride 66a (E)-tert-Butyl3-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamido)piperidine-1-carboxylate

The title compound, obtained as a yellow oil (103 mg, 27%), was preparedfrom the product of Example 1a by following the procedure of Example 3except tert-butyl 3-aminopiperidine-1-carboxylate was substituted formethylamine hydrochloride. R_(f)=0.20 in (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.31 (d, J=15.2, 1H), 5.69 (d, J=15.2 Hz, 1H),4.07 (s, 1H), 3.49-3.28 (m, 4H), 2.04-2.01 (m, 2H), 1.89 (s, 1H), 1.73(s, 3H), 1.71-1.58 (m, 5H), 1.55-1.46 (m, 11H), 1.05 (s, 6H) ppm; Massspectrum (ESI+ve) m/z 377.0 (MH⁺).

66b(E)-N-(Piperidin-3-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamidehydrochloride

The title compound, obtained as a pale brown amorphous solid (85 mg,99%), by following the procedure of Example 49b. R_(f)=0.1 (90:10)dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H-NMR (400MHz, CDCl₃) δ 9.61-9.53 (m, 2H), 8.03 (s, 1H), 7.33 (d, J=14.5 Hz, 1H),6.00 (d, J=14.5 Hz, 1H), 4.51 (s, 1H), 3.30-3.01 (m, 4H), 2.01-1.98 (m,1H), 1.74 (s, 2H), 1.58 (s, 3H), 1.45-1.43 (m, 2H), 1.24-1.22 (m, 2H),1.04 (s, 6H); Mass spectrum (ESI+ve) m/z 277 (MH⁺).

Example 67(E)-N-Methyl-N-(piperidin-3-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamidehydrochloride 67a (E)-tert-Butyl4-(N-methyl-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamido)piperidine-1-carboxylate

The title compound, obtained as a yellow oil (195 mg, 49%), was preparedfrom the product of Example 1a by following the procedure of Example 3except tert-butyl 4-(methylamino)piperidine-1-carboxylate wassubstituted for methylamine hydrochloride. R_(f)=0.25 in (25:75 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.35 (d, J=14.4, 1H), 6.34(d, J=14.4 Hz, 1H), 4.71 (s, 1H), 4.21 (s, 2H), 2.89 (s, 3H), 2.87-2.66(m, 2H), 2.04-2.02 (m, 2H), 1.75 (s, 3H), 1.65-1.58 (m, 6H), 1.48-1.46(m, 11H), 1.05 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 391.0 (MH⁺).

67b(E)-N-Methyl-N-(piperidin-3-yl)-3-(2,6,6-trimethycyclohex-1-en-1-yl)acrylamidehydrochloride

The title compound, obtained as a yellow film (160 mg, 99%), wasprepared by following the procedure of Example 49b. R_(f)=0.3 (90:10dichloromethane:methanol+0.1% (v/v) ammonium hydroxide); ¹H-NMR (400MHz, CDCl₃) δ 7.30 (d, J=15.0, 1H), 6.34 (d, J=15.0 Hz, 1H), 4.66 (s,1H), 3.52-3.49 (m, 2H), 3.17-3.11 (m, 2H), 3.03-2.94 (m, 3H), 2.10-2.07(m, 4H), 1.91-1.87 (m, 2H), 1.78 (s, 3H), 1.67-1.64 (m, 2H), 1.52-1.49(m, 2H), 1.07 (s, 6H), 0.89-0.85 (m, 2H); Mass spectrum (ESI+ve) m/z 291(MH⁺).

Example 68(E)-1-(1,1-Dioxidothiomorpholino)-3-(2,6,6-trimethylcyclohex-1-eyl)prop-2-en-1-one

The title compound, obtained as a pale yellow waxy solid (28.2 mg, 18%),was prepared from the product of Example 1a by following the procedureof Example 3 except thiomorpholine 1,1-dioxide was substituted formethylamine hydrochloride. R_(f)=0.10 (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.42 (d, J=15.5, 1H), 6.20 (d, J=15.5 Hz, 1H),4.10-4.07 (m, 4H), 3.10-2.98 (m, 4H), 2.07-1.98 (m, 2H), 1.75 (s, 3H),1.61 (t, J=6.0 Hz, 2H), 1.48 (t, J=6.0 Hz, 2H), 1.05 (s, 6H); Massspectrum (ESI+ve) m/z 312 (MH⁺).

Example 69(E)-1-Thiomorpholino-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one

The title compound, obtained as a clear oil (104 mg, 73%), was preparedfrom the product of Example 1a by following the procedure of Example 3except thiomorpholine was substituted for methylamine hydrochloride.R_(f)=0.20 (10:90 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 7.32(d, J=15.5, 1H), 6.18 (d, J=15.5 Hz, 1H), 3.95-3.85 (m, 4H), 2.67-2.65(m, 4H), 2.03 (t, J=6.0 Hz, 2H), 1.74 (s, 3H), 1.61 (t, J=6.0 Hz, 2H),1.46 (t, J=6.0 Hz, 2H), 1.05 (s, 6H); Mass spectrum (ESI+ve) m/z 280(MH⁺).

Example 70(E)-1-(4,4-Difluoropiperidin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one

The title compound, obtained as a white solid (155 mg, 81%), wasprepared by following the procedure of Example 3, except4,4-difluoropiperidine was substituted for methylamine hydrochloride.Mp=67.3-70.7° C.; R_(f)=0.44 (20:80 ethyl acetate:hexanes); ¹H-NMR (400MHz, CDCl₃) δ 7.37 (d, J=15.5 Hz, 1H), 6.24 (d, J=15.5 Hz, 1H), 3.75 (brm, 4H), 2.09-1.96 (m, 6H), 1.76 (s, 3H), 1.63 (m, 2H), 1.55-1.45 (m,2H), 1.07 (s, 6H); Mass spectrum (ESI+ve) m/z 298 (MH⁺).

Example 71 (±)-4-((E)-3-((1,6-anti)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide 71a (±)-(E)-Ethyl3-((1,6-anti)-2,2,6-trimethylcyclohexyl)acrylate

In a round bottom flask, sodium hydride (60% dispersion, 0.780 g, 19.4mmol) was suspended in hexanes (10 mL) and the solvent was decanted. Theresidue was suspended in anhydrous tetrahydrofuran (40 mL) and thereaction flask was charged with argon and cooled to 0′C. To the stirredslurry was added triethylphosphonoacetate (3.24 mL, 3.63 g, 16.2 mmol)dropwise as to prevent build-up of the foaming reaction mixture. Thereaction mixture was stirred for 30 minutes while warming to roomtemperature until a clear solution remained. To this stirred solutionwas added a solution of(1,6-anti)-2,2,6-trimethylcyclohexanecarbaldehyde (2.00 g, 12.9 mmol) inanhydrous tetrahydrofuran (10 mL). The reaction was heated to reflux andstirred for 18 hours.

Upon cooling to room temperature, the reaction mixture was transferredto a separatory funnel and diluted with ethyl acetate (300 mL). Theorganic phase was extracted with a 50% saturated solution of ammoniumchloride (2×100 mL) and then washed with brine (100 mL), dried overmagnesium sulfate, filtered and concentrated in vacuo to give a browncrude oil (˜5 g). The title compound was purified by flash columnchromatography (solvent gradient of 1-10% ethyl acetate:hexanes) toyield a pale brown oil (0.726 g, 28%). R_(f)=0.50 (5:95 ethylacetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 6.71 (dd, J=15.5, 10.0 Hz,1H), 5.74 (d, J=15.5 Hz, 1H), 4.16 (q, J=7.0 Hz, 2H), 1.76-1.65 (m, 1H),1.57-1.35 (m, 5H), 1.27 (t, J=7.0 Hz, 3H), 1.15 (m, 1H), 0.93-0.88 (m,1H), 0.86 (s, 3H), 0.80 (s, 3H), 0.72 (d, J=6 Hz, 3H); Mass spectrum(ESI+ve) m/z 225 (MH⁺).

71b (±)-(E)-3-((1,6-anti)-2,2,6-Trimethylcyclohexyl)acrylic acid

(E)-Ethyl 3-((1,6-anti)-2,2,6-trimethylcyclohexyl)acrylate (71a, 1.03 g,4.59 mmol) was dissolved in a 2:1 mixture of tetrahydrofuran (16 mL) andwater (8 mL) and lithium hydroxide (0.549 g, 22.9 mmol) was added to thesolution. The reaction mixture was heated to reflux and stirredovernight for 18 hours.

The reaction mixture was transferred to a separatory funnel and dilutedwith a 1M solution of sodium hydroxide (100 mL) and extracted withhexanes (50 mL). The aqueous phase was then acidified by addingconcentrated hydrochloric acid (˜16 mL) and then extracted withdichloromethane (3×50 mL). The combined organic layers were washed withbrine (50 mL), dried over magnesium sulfate, filtered and concentratedin vacuo to give a pale yellow solid (0.61 g, 67%). R_(f)=0.20 (25:75ethyl acetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 10.50 (br s, 1H), 6.86(dd, J=15.5, 10.0 Hz, 1H), 5.78 (d, J=15.5 Hz, 1H), 1.73 (m, 1H), 1.50(m, 5H), 1.25-1.12 (m, 1H), 0.98-0.91 (m, 1H), 0.89 (s, 3H), 0.83 (s,3H), 0.75 (d, J=6.0 Hz, 3H); Mass spectrum (ESI+ve) m/z 197 (MH⁺).

71c (±)-tert-Butyl4-((E)-3-((1,6-anti)-2,2,6-Trimethylcyclohexyl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a white solid (0.40 g, 95%), wasprepared from the product of Example 71b by following the procedure ofExample 3 except tert-butyl piperazine-1-carboxylate was substituted formethylamine hydrochloride. R_(f)=0.40 in (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 6.69 (dd, J=15.0, 10.0 Hz, 1H), 6.16 (d,J=15.0 Hz, 1H), 3.73-3.39 (m, 8H), 1.74 (m, 1H), 1.60-1.39 (m, 15H),1.22-1.12 (m, 1H), 0.90 (s, 3H), 0.83 (s, 3H), 0.77 (d, J=6.0 Hz, 3H);Mass spectrum (ESI+ve) m/z 365 (MH⁺).

71d4-((E)-3-((1,6-anti)-2,2,6-Trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide

To a solution of tert-butyl4-((E)-3-((1,6-anti)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxylate(71c, 0.35 g, 0.96 mmol) in dichloromethane (10 mL) was added dropwise a4.0 M solution of hydrochloric acid in 1,4-dioxane (1.2 mL, 4.8 mmol).The reaction mixture was stirred at room temperature for 18 hours thenconcentrated in vacuo.

The crude oil was dissolved in dichloromethane (10 mL) and potassiumcarbonate (0.67 g, 4.8 mmol) and trimethylsilyl isocyanate (1.3 mL, 9.6mmol) were added to the reaction mixture at room temperature. Thereaction mixture was stirred at room temperature for 18 hours.

The reaction mixture was poured into a saturated aqueous solution ofammonium chloride (10 mL) and was extracted with dichloromethane (3×10mL). The combined organic phases were dried over sodium sulfate and theconcentrated in vacuo. The product was purified by flash columnchromatography to yield the title compound as a white solid (0.205 mg,69%). R_(f)=0.20 in (5:95 methanol:chloroform); ¹H-NMR (400 MHz, CDCl₃)δ 6.68 (dd, J=15.0, 10.0 Hz, 1H), 6.13 (d, J=15.0 Hz, 1H), 4.76 (br s,2H), 3.52 (m, 8H), 1.91 (s, 1H), 1.72 (m, 1H), 1.50 (m, 5H), 1.15 (m,1H), 0.87 (s, 3H), 0.81 (s, 3H), 0.74 (d, J=6.0 Hz, 3H); Mass spectrum(ESI+ve) m/z 308 (MH⁺).

Example 72 (−)-4-((E)-3-((1R,6R)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide 72a (−)-(E)-Ethyl3-((1R,6R)-2,2,6-trimethylcyclohexyl)acrylate

The title compound, obtained as a pale brown oil (1.3 g, 18%), wasprepared by following the procedure of Example 71a except(1R,6R)-2,2,6-trimethylcyclohexanecarbaldehyde was substituted for(1,6-anti)-2,2,6-trimethylcyclohexanecarbaldehyde. [α]_(D) ²³=−15.3°(c=0.25, CHCl₃); R_(f)=0.50 (5:95 ethyl acetate:hexanes); ¹H NMR (400MHz, CDCl₃) δ 6.71 (dd, J=15.5, 10.0 Hz, 1H), 5.74 (d, J=15.5 Hz, 1H),4.16 (q, J=7.0 Hz, 2H), 1.76-1.65 (m, 1H), 1.57-1.35 (m, 5H), 1.27 (t,J=7.0 Hz, 3H), 1.15 (m, 1H), 0.93-0.88 (m, 1H), 0.86 (s, 3H), 0.80 (s,3H), 0.72 (d, J=6 Hz, 3H); Mass spectrum (ESI+ve) m/z 225 (MH⁺).

72b (−)-(E)-3-((1R,6R)-2,2,6-Trimethylcyclohexyl)acrylic acid

The title compound, obtained as a pale yellow solid (1.02 g, 91%), wasprepared from the product of 72 a by following the procedure of Example60c. [α]_(D) ²³=−20.0° (c=0.25, CHCl₃); R_(f)=0.20 (25:75 ethylacetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 10.50 (br s, 1H), 6.86 (dd,J=15.5, 10.0 Hz, 1H), 5.78 (d, J=15.5 Hz, 1H), 1.73 (m, 1H), 1.50 (m,5H), 1.25-1.12 (m, 1H), 0.98-0.91 (m, 1H), 0.89 (s, 3H), 0.83 (s, 3H),0.75 (d, J=6.0 Hz, 3H); Mass spectrum. (ESI+ve) m/z 197 (MH⁺).

72c (−)-tert-Butyl 4-((E)-3-((1R,6R)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a white solid (0.16 g, 86%), wasprepared from the product of Example 72b by following the procedure ofExample 3 except tert-butyl piperazine-1-carboxylate was substituted formethylamine hydrochloride. R_(f)=0.40 in (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 6.69 (dd, J=15.0, 10.0 Hz, 1H), 6.16 (d,J=15.0 Hz, 1H), 3.73-3.39 (m, 8H), 1.74 (m, 1H), 1.60-1.39 (m, 15H),1.22-1.12 (m, 1H), 0.90 (s, 3H), 0.83 (s, 3H), 0.77 (d, J=6.0 Hz, 3H);Mass spectrum (ESI+ve) m/z 365 (MH⁺).

72d(−)-4-((E)-3-((1R,6R)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a white solid (85 mg, 63%), was preparedfrom the product of example 72c by following the procedure of Example71d. [α]_(D) ²³=−22.4° (c=0.25, CHCl₃); ¹H-NMR (400 MHz, CDCl₃) δ 6.68(dd, J=15.0, 10.0 Hz, 1H), 6.13 (d, J=15.0 Hz, 1H), 4.76 (br s, 2H),3.52 (m, 8H), 1.91 (s, 1H), 1.72 (m, 1H), 1.50 (m, 5H), 1.15 (m, 1H),0.87 (s, 3H), 0.81 (s, 3H), 0.74 (d, J=6.0 Hz, 3H); Mass spectrum(ESI+ve) m/z 308 (MH⁺).

Example 73 (+)-4-((E)-3-((1 S,6S)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide 73a (+)-(E)-Ethyl 3-((1S,6S)-2,2,6-trimethylcyclohexyl)acrylate

The title compound, obtained as a pale brown oil (1.4 g, 28%), wasprepared by following the procedure of Example 71a except(1S,6S)-2,2,6-trimethylcyclohexanecarbaldehyde was substituted for(1,6-anti)-2,2,6-trimethylcyclohexanecarbaldehyde. [α]_(D) ²³=+16.8°(c=0.25, CHCl₃); R_(f)=0.50 (5:95 ethyl acetate:hexanes); ¹H NMR (400MHz, CDCl₃) δ 6.71 (dd, J=15.5, 10.0 Hz, 1H), 5.74 (d, J=15.5 Hz, 1H),4.16 (q, J=7.0 Hz, 2H), 1.76-1.65 (m, 1H), 1.57-1.35 (m, 5H), 1.27 (t,J=7.0 Hz, 3H), 1.15 (m, 1H), 0.93-0.88 (m, 1H), 0.86 (s, 3H), 0.80 (s,3H), 0.72 (d, J=6 Hz, 3H); Mass spectrum (ESI+ve) m/z 225 (MH⁺).

73b (+)-(E)-3-((1 S,6S)-2,2,6-Trimethylcyclohexyl)acrylic acid

The title compound, obtained as a pale yellow solid (0.61 g, 67%), wasprepared from the product of 73a by following the procedure of Example60c. [α]_(D) ²³=+21.6° (c=0.25, CHCl₃); R_(f)=0.20 (25:75 ethylacetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 10.50 (br s, 1H), 6.86 (dd,J=15.5, 10.0 Hz, 1H), 5.78 (d, J=15.5 Hz, 1H), 1.73 (m, 1H), 1.50 (m,5H), 1.25-1.12 (m, 1H), 0.98-0.91 (m, 1H), 0.89 (s, 3H), 0.83 (s, 3H),0.75 (d, J=6.0 Hz, 3H); Mass spectrum (ESI+ve) m/z 197 (MH⁺).

73c (+)-tert-Butyl 4-((E)-3-((1S,6S)-2,2,6-trimethylcyclohexyl)acryloyl) piperazine-1-carboxylate

The title compound, obtained as a white solid (0.40 g, 98%), wasprepared from the product of Example 73b by following the procedure ofExample 3 except tert-butyl piperazine-1-carboxylate was substituted formethylamine hydrochloride. R_(f)=0.40 in (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 6.69 (dd, J=15.0, 10.0 Hz, 1H), 6.16 (d,J=15.0 Hz, 1H), 3.73-3.39 (m, 8H), 1.74 (m, 1H), 1.60-1.39 (m, 15H),1.22-1.12 (m, 1H), 0.90 (s, 3H), 0.83 (s, 3H), 0.77 (d, J=6.0 Hz, 3H);Mass spectrum (ESI+ve) m/z 365 (MH⁺).

73d(+)-4-((E)-3-((1S,6S)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a white solid (0.21 g, 69%), wasprepared from the product of example 73c by following the procedure ofExample 71d. [O]_(D) ²³=+19.6° (c=0.25, CHCl₃); ¹H-NMR (400 MHz, CDCl₃)δ 6.68 (dd, J=15.0, 10.0 Hz, 1H), 6.13 (d, J=15.0 Hz, 1H), 4.76 (br s,2H), 3.52 (m, 8H), 1.91 (s, 1H), 1.72 (m, 1H), 1.50 (m, 5H), 1.15 (m,1H), 0.87 (s, 3H), 0.81 (s, 3H), 0.74 (d, J=6.0 Hz, 3H); Mass spectrum(ESI+ve) m/z 308 (MH⁺).

Example 74(E)-1-Morpholino-3-((1R,6R)-2,2,6-trimethylcyclohexyl)prop-2-en-1-one

The title compound, obtained as a clear viscous oil (57.0 mg, 86%), wasprepared from the product of Example 72b by following the procedure ofExample 3 except morpholine was substituted for methylaminehydrochloride. [α]_(D) ²³=−21.6° (c=0.32, chloroform); R_(f)=0.30 in(10:90 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 6.81 (dd,J=15.0, 8.5 Hz, 1H), 6.12 (d, J=15.0 Hz, 1H), 3.69-3.56 (m, 8H),1.75-1.71 (m, 2H), 1.51-1.35 (m, 5H), 0.88-0.87 (m, 4H), 0.82 (s, 3H),0.75 (d, J=6.0 Hz, 3H); Mass spectrum (ESI+ve) m/z 266 (MH⁺).

Example 75 (E)-1-Thiomorpholino-3-((1R,6R)-2,2,6-trimethylcyclohexyl)prop-2-en-1-one

The title compound, obtained as a clear oil (38.0 mg, 54%), was preparedfrom the product of Example 72b by following the procedure of Example 3except thiomorpholine was substituted for methylamine hydrochloride.[α]_(D) ²³=−20.4° (c=0.32, chloroform); R_(f)=0.40 in (10:90 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 6.63 (dd, J=15.0, 8.5 Hz,1H), 6.18 (d, J=15.0 Hz, 1H), 3.90-3.82 (m, 4H), 2.62 (s, 4H), 1.73-1.70(m, 1H), 1.49-1.47 (m, 5H), 1.13-1.12 (m, 1H), 0.87-0.84 (m, 4H), 0.89(s, 3H), 0.74 (d, J=6.0 Hz, 3H); Mass spectrum (ESI+ve) m/z 282.1 (MH⁺).

Example 76 (E)-4-(3-(2,5,6,6-tetramethylcyclohex-2-en-1-yl)acryloyl)piperazine-1-carboxamide 76a(E)-3-(2,5,6,6-tetramethylcyclohex-2-en-1-yl)acrylic acid

The title compound, obtained as a clear viscous oil (1.90 g, 50%), wasprepared from irone (≦90% α:β isomers) by following the procedure ofExample 1. R_(f)=0.6 (25:75 ethyl acetate:hexanes+0.1% (v/v) aceticacid); ¹H-NMR (400 MHz, CDCl₃) δ 11.71 (br s, 1H), 7.02-6.91 (m, 1H),5.83 (q, J=15.0 Hz, 1H), 5.49 (d, J=17.5 Hz, 1H), 2.57 (d, J=11.0 Hz,0.43H), 2.29 (d, J=9.5 Hz, 0.57H), 2.03-1.91 (m, 1H), 1.69-1.67 (m, 2H),1.54 (d, J=16.5 Hz, 1H) 1.10 (br s, 0.43H), 0.87-0.81 (m, 8H), 0.70 (s,1H); Mass spectrum (ESI−ve) m/z 207 (MH⁻).

76b(E)-4-(3-(2,5,6,6-tetramethylcyclohex-2-en-1-yl)acryloyl)piperazine-1-carboxamide

To a solution of (E)-3-(2,5,6,6-tetramethylcyclohex-2-en-1-yl)acrylicacid (76a, 100 mg, 0.480 mmol) in acetonitrile (5.0 mL) was added2-(7-aza-1H-benzotriazole-1-yl)-1, 1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 182 mg, 0.480 mmol). The solution was stirredat room temperature for 30 minutes then diisopropylethylamine (62.0 mg,0.480 mmol) and tert-butyl piperazine-1-carboxylate (89.4 mg, 0.480mmol) was added to the reaction mixture. The reaction was then stirredat 40° C. for 4 hours.

The reaction was quenched with a 1M solution of hydrochloric acid (2 mL)and the biphasic mixture was separated. The organic layer wasconcentrated in vacuo (40° C.) and the crude material loaded on tosilica gel for purification via flash column chromatography running anisocratic eluent of 30% ethyl acetate in hexanes. The intermediate wasisolated as a white solid (120 mg, 66%).

The intermediate was dissolved in dichloromethane (10 mL) was addeddropwise a 4.0 M solution of hydrochloric acid in 1,4-dioxane (1.2 mL,4.8 mmol). The reaction mixture was stirred at room temperature for 18hours then concentrated in vacuo.

The crude oil was dissolved in dichloromethane (10 mL) and potassiumcarbonate (0.67 g, 4.8 mmol) and trimethylsilyl isocyanate (1.3 mL, 9.6mmol) were added to the reaction mixture at room temperature. Thereaction mixture was stirred at room temperature for 18 hours.

The reaction mixture was poured into a saturated aqueous solution ofammonium chloride (10 mL) and was extracted with dichloromethane (3×10mL). The combined organic phases were dried over sodium sulfate and theconcentrated in vacuo. The product was purified by preparative platethin layer chromatography to yield the title compound as a clear oil(13.0 mg, 11%). R_(f)=0.40 (5:95 methanol:chloroform); ¹H-NMR (400 MHz,CDCl₃) δ 6.85-6.74 (m, 1H), 6.14 (m, 1H), 5.46 (d, J=18.0 Hz, 1H), 4.85(s, 2H), 3.72-3.41 (m, 8H), 2.26 (d, J=11.0 Hz, 0.44H), 2.03 (d, J=9.5Hz, 0.57H), 2.04-1.90 (m, 1H), 1.69-1.64 (m, 1H), 1.54 (d, J=15.0 Hz,3H), 0.87-0.80 (m, 8H), 0.71 (s, 1H); Mass spectrum (ESI+ve) m/z 320(MH⁺).

Example 774-((E)-3-((1R,6S)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamideand 4-((E)-3-((1 S,6R)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide 77a(±)-1,6-syn-3-(2,2,6-trimethylcyclohexyl)propanoic acid

The title compound, obtained as a clear viscous oil (316 mg, 16%), wasprepared from tetrahydroionone (2.00 g, 10.1 mmol) by following theprocedure of Example 1. R_(f)=0.30 (10:90 ethyl acetate:hexanes+0.1%(v/v) acetic acid); ¹H-NMR (400 MHz, CDCl₃) δ 10.19 (br s, 1H), 2.33 (t,J=8.5 Hz, 2H), 1.93-1.90 (m, 1H), 1.62-1.58 (m, 2H), 1.46-1.44 (m, 3H),1.33-1.29 (m, 2H), 1.11-1.09 (m, 2H), 0.95 (s, 3H), 0.94 (s, 6H); Massspectrum (ESI−ve) m/z 197 (MH⁻).

77b4-((E)-3-((1R,6S)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamideand 4-((E)-3-((1 S,6R)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a white film (17.0 mg, 36%), wasprepared from the product of Example 77a by following the procedure ofExample 76b. R_(f)=0.50 (5:95 methanol:chloroform); ¹H-NMR (400 MHz,CDCl₃) δ 4.61 (s, 2H), 3.66 (t, J=5.0 Hz, 2H), 3.49 (s, 4H), 3.36 (t,J=5.0 Hz, 2H), 2.29 (t, J=8.5 Hz, 2H), 1.93-1.89 (m, 1H), 1.32-1.29 (m,5H), 1.16-1.43 (m, 3H), 1.10-1.02 (m, 3H), 0.95 (s, 3H), 0.89 (S, 3H),0.87 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 310 (MH⁺).

Example 78(E)-4-(3-(2,6,6-trimethylcyclohex-2-en-1-yl)acryloyl)piperazine-1-carboxamide78a (E)-3-(2,6,6-trimethylcyclohex-2-on-1-yl)acrylic acid

The title compound, obtained as a clear viscous oil (56 mg, 3%), wasprepared from a-ionone (2.00 g, 10.3 mmol) by following the procedure ofExample 1. R_(f)=0.2 (10:90 ethyl acetate:hexanes+0.1% (v/v) aceticacid); ¹H-NMR (400 MHz, CDCl₃) δ 11.33 (br s, 1H), 6.95-6.88 (m, 1H),5.81 (d, J=15.0 Hz, 1H), 5.49 (s, 1H), 2.31-2.29 (m, 1H), 2.04 (s, 2H),1.50 (s, 3H), 1.48-1.43 (m, 1H), 1.22-1.18 (m, 1H), 0.92 (s, 3H), 0.89(s, 3H); Mass spectrum (ESI−ve) m/z 193 (MH⁻).

78b(E)-4-(3-(2,6,6-trimethylcyclohex-2-en-1-yl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a white solid (22.0 mg, 36%), wasprepared from the product of Example 78a by following the procedure ofExample 76b. R_(f)=0.50 (5:95 methanol:chloroform); ¹H-NMR (400 MHz,CDCl₃) δ 6.75 (dd, J=14.5, 9.5 Hz, 1H), 6.15 (d, J=14.5 Hz, 1H), 5.47(s, 1H), 4.72 (s, 2H), 3.59-3.40 (m, 8H), 2.27 (d, J=9.5 Hz, 1H), 2.01(br s, 2H), 1.56 (s, 3H), 1.50-1.42 (m, 1H), 1.24-1.18 (m, 1H), 0.97 (s,3H), 0.84 (s, 3H); Mass spectrum (ESI+ve) m/z 306 (MH⁺).

Example 79(±)-4-((E)-3-(1,3,3-Trimethyl-7-oxabicyclo[4.1.0]heptan-2-yl)acryloyl)piperazine-1-carboxamide 79a(±)-(E)-3-(1,3,3-trimethyl-7-oxabicyclo[4.1.0]heptan-2-yl)acrylic acid

The title compound, obtained as a white solid (616 mg, 31%), wasprepared from4-(1,3,3-trimethyl-7-oxabicyclo[4.1.0]hept-2-yl)-3-buten-2-one (290%cis:trans isomers) (2.00 g, 9.60 mmol) by following the procedure ofExample 1. Mp=122.2-130.6° C.; R_(f)=0.20 (10:90 ethylacetate:hexanes+0.1% (v/v) acetic acid); ¹H-NMR (400 MHz, CDCl₃) δ 11.84(br s, 1H), 7.07-6.92 (m, 1H), 5.91 (d, J=15.5 Hz, 1H), 3.07 (s, 1H),2.10 (d, J=11.0 Hz, 1H), 1.97-1.94 (m, 1H), 1.86-1.73 (m, 1H), 1.49-1.39(m, 1H), 1.39 (s, 3H), 1.05-0.98 (m, 11H) 0.93 (s, 3H), 0.77 (s, 3H);Mass spectrum (ESI−ve) m/z 209 (MH⁻).

79b(±)-4-((E)-3-(1,3,3-Trimethyl-7-oxabicyclo[4.1.0]heptan-2-yl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as an off-white solid (19.9 mg, 44%), wasprepared from the product of Example 79a by following the procedure ofExample 76b. R_(f)=0.30 (5:95 methanol:chloroform); ¹H-NMR (400 MHz,CDCl₃) δ 6.75 (dd, J=15.0, 10.0 Hz, 1H), 6.27 (d, J=15.0 Hz, 1H), 4.71(s, 2H), 3.70-3.43 (m, 8H), 3.05 (s, 1H), 2.08 (d, J=10.0 Hz, 1H),1.96-87 (m, 2H), 1.43-1.41 (m, 1H), 1.25 (s, 3H), 0.97-0.90 (m, 1H),0.91 (s, 3H), 0.86 (s, 3H); Mass spectrum (ESI+ve) m/z 322 (MH⁺).

Example 804-(3-((1R,6S)-2,2,6-trimethylcyclohexyl)propanoyl)piperazine-1-carboxamide

4-((E)-3-((1S,6S)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide (73d,50.0 mg, 0.16 mmol) was dissolved in anhydrous methanol (5.0 mL) at roomtemperature under argon to which 10% palladium on carbon (3.00 mg, 5.00mmol) was added and allowed to stir vigorously. The reaction vessel wasevacuated under vacuum and then charged with hydrogen gas, the reactionmixture was left to stir at room temperature for 2 days.

The reaction mixture was filtered through Celite and the solvent wasremoved in vacuo to yield a white solid (50.0 mg, 99%). ¹H-NMR (400 MHz,CDCl₃) δ 4.67 (br s, 2H), 3.65-3.63 (m, 2H), 3.48 (br s, 4H), 3.67-3.45(m, 2H), 2.40-2.26 (m, 2H), 1.75-1.60 (m, 2H), 1.44-1.33 (m, 5H),1.17-1.09 (m, 1H), 0.93-0.87 (m, 7H), 0.80 (s, 3H), 0.59-0.57 (m, 1H);Mass spectrum (ESI+ve) m/z 310 (MH⁺).

Example 814-(3-((1S,6R)-2,2,6-trimethylcyclohexyl)propanoyl)piperazine-1-carboxamide

4-((E)-3-((1R,6R)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide(72d, 28.0 mg, 0.09 mmol) was dissolved in anhydrous methanol (5.00 mL)at room temperature under argon to which 10% palladium on carbon (3.00mg, 5.00 mmol) was added and allowed to stir vigorously. The reactionvessel was evacuated under vacuum and then charged with hydrogen gas,the reaction mixture was left to stir at room temperature for 2 days.

The reaction mixture was filtered through Celite and the solvent wasremoved in vacuo to yield a white solid (28.0 mg, 99%). ¹H-NMR (400 MHz,CDCl₃) δ 4.67 (br s, 2H), 3.65-3.63 (m, 2H), 3.48 (br s, 4H), 3.67-3.45(m, 2H), 2.40-2.26 (m, 2H), 1.75-1.60 (m, 2H), 1.44-1.33 (m, 5H),1.17-1.09 (m, 1H), 0.93-0.87 (m, 7H), 0.80 (s, 3H), 0.59-0.57 (m, 1H);Mass spectrum (ESI+ve) m/z 310 (MH⁺).

Example 82(E)-4-(3-(2-chloro-3-hydroxy-2,6,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxamide

To a solution of(±)-(E)-3-(1,3,3-trimethyl-7-oxabicyclo[4.1.0]heptan-2-yl)acrylic acid(79a, 100 mg, 0.476 mmol) in acetonitrile (5.0 mL) was added2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 181 mg, 0.476 mmol). The solution was stirredat room temperature for 30 minutes then diisopropylethylamine (61.5 mg,0.476 mmol) and tert-butyl piperazine-1-carboxylate (88.6 mg, 0.476mmol) was added to the reaction mixture. The reaction was then stirredat 40° C. for 4 hours.

The reaction was quenched with a 1M solution of hydrochloric acid (2 mL)and the biphasic mixture was separated. The organic layer wasconcentrated in vacuo (40° C.) and the crude material loaded on tosilica gel for purification via flash column chromatography running anisocratic eluent of 30% ethyl acetate in hexanes. The intermediate wasisolated as a white solid (135 mg, 75%).

The intermediate was dissolved in dichloromethane (10 mL) was addeddropwise a 4.0 M solution of hydrochloric acid in 1,4-dioxane (1.2 mL,4.8 mmol). The reaction mixture was stirred at room temperature for 18hours then concentrated in vacuo.

The crude oil was dissolved in dichloromethane (10 mL) and potassiumcarbonate (0.67 g, 4.8 mmol) and trimethylsilyl isocyanate (1.3 mL, 9.6mmol) were added to the reaction mixture at room temperature. Thereaction mixture was stirred at room temperature for 18 hours.

The reaction mixture was poured into a saturated aqueous solution ofammonium chloride (10 mL) and was extracted with dichloromethane (3×10mL). The combined organic phases were dried over sodium sulfate and theconcentrated in vacuo. The product was purified by preparative platethin layer chromatography to yield the title compound as a white solid(19.0 mg, 15%). R_(f)=0.30 (10:90 methanol:chloroform); ¹H-NMR (400 MHz,CD₃OD) δ 6.97 (dd, J=15.5, 11.0 Hz, 1H), 6.46 (d, J=15.5 Hz, 1H), 3.99(s, 1H), 3.68-3.66 (m, 4H), 3.44-3.42 (m, 4H), 2.57-2.54 (m, 1H),2.29-2.26 (m, 1H), 1.89-1.82 (m, 1H), 1.70-1.66 (m, 1H), 1.29-1.26 (m,1H), 1.23-1.20 (m, 1H), 1.13 (s, 3H), 1.04 (s, 3H), 0.81 (s, 3H); Massspectrum (ESI+ve) m/z 358 [³⁵Cl, ³⁵Cl], 359.0 [³⁵Cl, ³⁷Cl], 360.1 [³⁷Cl,³⁷Cl] (MH⁺).

Example 83(E)-1-Morpholino-3-((1S,6S)-2,2,6-trimethylcyclohexyl)prop-2-en-1-one

The title compound, obtained as a clear oil (62.1 mg, 74%), was preparedfrom the product of Example 71b by following the procedure of Example 3except morpholine was substituted for methylamine hydrochloride. [α]_(D)²³=+22.8° (c=0.25, chloroform); R_(f)=0.50 in (40:60 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 6.81 (dd, J=15.0, 8.5 Hz,1H), 6.12 (d, J=15.0 Hz, 1H), 3.69-3.56 (m, 8H), 1.75-1.71 (m, 2H),1.51-1.35 (m, 5H), 0.88-0.87 (m, 4H), 0.82 (s, 3H), 0.75 (d, J=6.0 Hz,3H); Mass spectrum (ESI+ve) m/z 266 (MH⁺).

Example 84(E)-4-(3-(2,6,6-Trimethylcyclohex-14-en-1-yl)acryloyl)piperazin-2-one

The title compound, obtained as a clear oil (165 mg, 60%), was preparedfrom the product of Example 1 by following the procedure of Example 3except piperazin-2-one was substituted for methylamine hydrochloride.R_(f)=0.36 in (15:85 ethyl acetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ7.42 (d, J=15.0 Hz, 1H), 6.98 (d, J=2.0 Hz, 1H), 6.15 (d, J=15.0 Hz,1H), 4.23 (s, 2H), 3.83 (m, 2H), 3.43 (m, 2H), 2.03 (m, 2H), 1.84-1.37(m, 7H), 1.04 (s, 6H); Mass spectrum (ESI+ve) m/z 277 (MH⁺).

Example 85(E)-1-(1,4-Dioxa-8-azaspiro[4.5]decan-8-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one

The title compound, obtained as a clear oil (295 mg, 92%), was preparedfrom the product of Example 1 by following the procedure of Example 3except 1,4-dioxa-8-azaspiro[4.5]decane was substituted for methylaminehydrochloride. R_(f)=0.2 in (40:60 ethyl acetate:hexanes); ¹H-NMR (400MHz, CDCl₃) δ 7.30 (d, J=15.0 Hz, 1H), 6.23 (d, J=15.0 Hz, 1H), 3.98 (s,4H), 3.81-3.53 (m, 4H), 2.03 (t, J=6.0 Hz, 1H), 1.73 (s, 3H), 1.72-1.53(m, 2H), 1.49-1.44 (m, 2H), 1.04 (s, 6H); Mass spectrum (ESI+ve) m/z 320(MH⁺).

Example 86 (E)-Ethyl 1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperidine-4-carboxylate

The title compound, obtained as a clear oil (286 mg, 83%), was preparedfrom the product of Example 1 by following the procedure of Example 3except ethyl piperidine-4-carboxylate was substituted for methylaminehydrochloride. R_(f)=0.5 in (50:50 ethyl acetate:hexanes); ¹H-NMR (400MHz, CDCl₃) δ 7.28 (d, J=15.0 Hz, 1H), 6.19 (d, J=15.0 Hz, 1H), 4.45 (brs, 1H), 4.13 (q, J=7.0 Hz, 2H), 3.94 (br s, 1H), 3.01 (m, 2H), 2.53 (tt,J=10.5, 4.0 Hz, 1H), 2.15-1.84 (m, 4H), 1.72 (s, 3H), 1.63 (m, 4H), 1.45(m, 2H), 1.24 (t, J=7.0 Hz, 3H), 1.03 (s, 6H); Mass spectrum (ESI+ve)m/z 334 (MH⁺).

Example 87(E)-4-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-sulfonamide

The title compound, obtained as a white solid (41.0 mg, 58%), wasprepared from the product of Example 1 by following the procedure ofExample 3 except piperazine-1-sulfonamide was substituted formethylamine hydrochloride. R_(f)=0.6 in (10:90 methanol:chloroform);Mp=181-183° C.; ¹H-NMR (400 MHz, CDCl₃) δ 7.44-7.32 (d, J=15.0 Hz, 1H),6.25-6.14 (d, J=15.0 Hz, 1H), 4.46 (br s, 2H), 3.75 (m, 4H), 3.21 (m,4H), 2.04 (t, J=6.05H, 2H), 1.75 (s, 3H), 1.62 (m, 2H), 1.48 (m, 2H),1.05 (s, 6H); Mass spectrum (ESI+ve) m/z 342 (MH⁺).

Example 88(E)-4-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carbaldehyde

Acetic anhydride (0.25 mL) was added to a solution of formic acid (1.25mL) at room temperature. To this stirred solution was added the productof Example 28b predissolved in formic acid (0.5 mL). The reaction wasstirred at room temperature for 16 hours.

The reaction mixture was then concentrated into silica gel and purifiedby flash column chromatography to yield the title compound as a yellowoil (92.0 mg, 46%). R_(f)=0.50 in (10:90 methanol:chloroform); ¹H-NMR(400 MHz, CDCl₃) δ 8.09 (s, 1H), 7.37 (d, J=15.5 Hz, 1H), 6.19 (d,J=15.5 Hz, 1H), 3.83-3.49 (m, 6H), 3.47-3.33 (m, 2H), 2.03 (t, J=6.0 Hz,2H), 1.73 (s, 3H), 1.60 (m, 2H), 1.52-1.36 (m, 2H), 1.04 (s, 6H); Massspectrum (ESI+ve) m/z 291 (MH⁺).

Example 89(E)-1-(4-(2-hydroxyethyl)piperazin-1-yl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)prop-2-en-1-one

The title compound, obtained as a clear oil (25.0 mg, 12%), was preparedfrom the product of example 28b by following the procedure of Example 7cexcept 2-bromoethanol was substituted for iodomethane. R_(f)=0.23 in(10:90 methanol:chloroform); ¹H-NMR (400 MHz, CDCl₃) δ 7.32 (d, J=15.5Hz, 1H), 6.19 (d, J=15.5 Hz, 1H), 3.81-3.44 (m, 6H), 2.72 (s, 1H),2.62-2.44 (m, 6H), 2.02 (t, J=6.0 Hz, 2H), 1.73 (s, 3H), 1.67-1.53 (m,2H), 1.50-1.37 (m, 2H), 1.03 (s, 6H); Mass spectrum (ESI+ve) m/z 307(MH⁺).

Example 90(±)-3,5-cis-Dimethyl-4-((E)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a yellow amorphous solid (242 mg, 86%),was prepared from the product of Example 1 by following the procedure ofExample 76b except tert-butyl cis-3,5-dimethylpiperazine-1-carboxylatewas substituted for tert-butyl piperazine-1-carboxylate. R_(f)=0.10 in(10:90 methanol:chloroform); Mp=152-155° C.; ¹H-NMR (400 MHz, CDCl₃) δ7.38 (d, J=15.5 Hz, 1H), 6.19 (d, J=15.5 Hz, 1H), 4.70 (s, 2H), 3.78 (m,4H), 3.15 (m Hz, 2H), 2.04 (t, J=6.0 Hz, 2H), 1.74 (s, 3H), 1.61 (m,2H), 1.52-1.41 (m, 2H), 1.33 (s, 3H), 1.32 (s, 3H), 1.09 (s, 6H); Massspectrum (ESI+ve) m/z 334 (MH⁺).

Example 91 (E)-4-(3-(2,2,6-trimethyibicyclo[4.1.0]heptan-1-yl)acryloyl)piperazine-1-carboxamide

Sodium borohydride (6.50 mg, 172 mmol) was added to a stirred solutionof β-cyclocitral (13.2 g, 86.0 mmol) in methanol (400 mL) at 0° C. underargon. The reaction was stirred at room temperature for 6 hours. Thereaction was quenched by adding water (400 mL) and extracted with ethylacetate (3×200 mL). The combined organic layers were washed with brine(600 mL), dried over magnesium sulfate, filtered and concentrated affordthe alcohol as a clear oil (13.5 g, quantitative).

In a dry round bottom flask diethyl zinc (1.0M in hexanes, 8.60 mL, 8.60mmol) was added to anhydrous diethyl ether (10 mL) at room temperature.To this solution was added dropwise methyleneiodide (0.71 mL, 8.85 mmol)and the reaction was stirred at room temperature for 15 minutesresulting in the formation of a white precipitate. The above preparedalcohol (910 mg, 5.90 mmol) was dissolved in diethyl ether (4.0 mL) andadded to the reaction mixture. The reaction was stirred a roomtemperature for 20 minutes and then heated to reflux for 16 hours. Thereaction was cooled to 0° C. and quenched with a saturated solution ofammonium chloride (2.0 mL). The biphasic reaction mixture wastransferred to a separatory funnel and diluted with diethyl ether (10mL) and the extracted with saturated ammonium chloride (20 mL) andwashed with brine (20 mL). The organic layer was dried over magnesiumsulfate, filtered and concentrated to give a brown oil (˜1.1 g). Thecyclopropanated alcohol was isolated by flash column chromatography(0-20% ethyl acetate in hexanes) as a clear oil (385 mg, 39%).

The cyclopropyl alcohol (371 mg, 2.20 mmol) was dissolved indichloromethane (34 mL) and Dess-Martin periodinane (1.03 g, 2.43 mmol)was added to the stirred solution followed by a drop of water (0.05 mL).The reaction was stirred at room temperature for 1 hour thenconcentrated to remove the dichloromethane. The residue was dissolved indiethyl ether (80 mL) and treated with a 1:1 (v/v) solution of 10%sodium thiosulfate (25 mL) and saturated sodium bicarbonate (25 mL) for30 minutes. The layers were separated and the organic phase was washedwith water (50 mL) and brine (50 mL), then dried over sodium sulfate,filtered and concentrated to give a crude solid. The aldehyde wasisolated by flash column chromatography (0-20% diethyl ether in hexanes)as a grey solid (260 mg, 71%).

The aldehyde was carried forward according to the procedure described inExamples 71a through 71d to furnish the title compound as a white solid(35.1 mg, 94%). R_(f)=0.17 in (5:95 methanol:chloroform); Mp=166-168°C.; ¹H-NMR (400 MHz, CD₃OD) δ 7.14 (dd, J=15.0, 3.0 Hz, 1H), 6.35 (dd,J=15.0, 3.0 Hz, 1H), 3.66 (m, 4H), 3.46 (m, 4H), 1.81-1.66 (m, 2H),1.62-1.25 (m, 3H), 1.25-1.10 (m, 6H), 0.99 (d, J=3.0 Hz, 3H), 0.91 (t,J=6.0 Hz, 3H), 0.67 (m, 2H); Mass spectrum (ESI+ve) m/z 320 (MH⁺).

Example 92 (E)-4-(3-(4-hydroxy-2,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

In 1 L Parr vessel reactor 4-oxo-isophorone (20.0 g, 130 mmol) wasdissolved in ethanol (200 mL) and Raney/Ni (0.1 eq) was added to thesolution. The vessel was charged with hydrogen gas to a pressure of 100psi. The reaction mixture was stirred at room temperature for 3 days,filtered through a pad of Celite, and the solvent was removed underreduced pressure to yield the product as clear oil, the crude wascarried forward into the next step of synthesis.

To a solution of crude 4-hydroxy-2,2,6-trimethylcyclohexan-1-one (20.2g, 130 mmol) and imidazole (35.3 g, 520 mmol) in dichloromethane (200mL) at 0° C., was added a solution of tert-butylchlorodimethylsilane(78.3 g, 260 mmol) in dichloromethane (200 mL). The reaction was stirredfor 16 h and then poured into water (100 mL) and extracted with hexane(3×150 mL). The organic layer was washed with water (5×100 mL), driedover magnesium sulfate and concentrated. The residue was purified byflash column chromatography (silica gel, 97:3 hexane:ethyl acetate) toafford 14.0 g (40%) of 4-(tert-butyldimethylsilyloxy)-2,2,6-trimethylcyclohexan-1-one as colorless oil.

To a solution of4-(tert-butyldimethylsilyloxy)-2,2,6-trimethylcyclohexan-1-one (7.00 g,26.0 mmol) in ethanol (50 mL) at 25° C., were added hydrazinemonohydrate (33.5 g, 670 mmol) and diisopropylethylamine (9.80 mL, 56.3mmol). After the mixture was stirred for 24 h at 100° C., the solventwas removed and the residue was taken in diethyl ether (30 mL) andwashed with brine (3×50 mL). The aqueous layers were extracted withdiethyl ether (4×50 mL), and the organic extracts were dried overmagnesium sulfate and concentrated.

To a solution of the residue in diethyl ether (30 mL) and1,5-diazabicyclo[4.3.0.]nonane (25.0 mL, 200 mmol) was added a solutionof iodine (9.90 g, 39.0 mmol) in diethyl ether (30 mL). After themixture was stirred for 15 min, an aqueous solution of saturated sodiumbicarbonate was added, the layers were separated, the organic layer wasdried over sodium sulfate, and the solvent was removed. A solution ofthe residue in benzene (60 mL) was treated with1,5-diazabicyclo[4.3.0.]nonane (25 mL). The mixture was stirred for 2.5h, then poured into diethyl ether (200 mL) and washed with aqueoussodium thiosulfate (3×30 mL), and the organic layer was dried andevaporated. The residue was purified by chromatography (silica gel, 5%ethyl acetate:hexanes) to afford 5.2 g (53%) oftert-butyldimethylsilyl-3,5,5-trimethyl-4-iodocyclohex-3-en-1-yl ether.

To a solution of the ether (0.80 g, 2.21 mmol) in N,N-dimethylforamide(10 mL) was added tetrakistriphenylphosphine palladium (0.240 g, 0.210mmol), and the mixture was degassed by the freeze-thaw method (threecycles). Methyl vinyl ketone (0.530 mL, 6.31 mmol) and triethylamine(0.880 mL, 6.31 mmol) were then added, and the reaction was heated to170° C. for 1 h using microwave irradiation. The mixture was dilutedwith diethyl ether (50 mL), washed with a 1% solution of hydrochloricacid, and extracted with Et₂O (3×25 mL). The combined organic layerswere washed with a saturated solution of aqueous sodium bicarbonate(3×25 mL) and dried over magnesium sulfate, and the solvent was removed.The resulting oil was purified by flash column chromatography (silicagel, 90:10 hexanes:ethyl acetate) affording 314 mg (42%) of[(E)-4-(tert-butyldimethylsilyloxy)-2,6,6-trimethylcyclohex-1-en-1-yl]but-3-en-2-oneas colorless oil.

The ketone was hydrolyzed to the carboxylic acid according to theprocedure outlined in Example 1, and the corresponding acrylic acid wascarried forward according to the procedure described in Examples 71bthrough 71d to furnish the title compound as a clear oil (10.1 mg, 30%).R_(f)=0.20 in (10:90 methanol:chloroform); ¹H-NMR (400 MHz, CDCl₃) δ6.19 (d, J=15.5 Hz, 1H), 4.66 (s, 2H), 4.08-3.91 (m, 1H), 3.84-3.34 (m,8H), 2.40 (dd, J=17.0, 5.5 Hz, 1H), 2.14-1.98 (m, 1H), 1.87-1.65 (m,5H), 1.47 (t, J=12.0 Hz, SH), 7.31 (d, J=15.5 Hz, 1H), 1.09 (s, 3H),1.08 (s, 3H); Mass spectrum (ESI+ve) m/z 322 (MH⁺).

Example 93 (±)-(E)-4-(3-(4-Methoxy-2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl) piperazine-1-carboxamide

To a solution of[4-(tert-butyldimethylsilyloxy)-2,6,6-trimethylcyclohex-1-en-1-yl]but-3-en-2one (230 mg, 0.680 mmol), prepared in Example 92, in tetrahydrofuran(2.0 mL) was added tetrabutylammonium fluoride (1.0 M in THF, 2.00 mL,2.00 mmol), and the mixture was stirred for 16 h at room temperature.The reaction was poured on to an aqueous solution of saturated sodiumbicarbonate, and extracted with diethyl ether (3×10 mL), and dried overmagnesium sulfate, and the solvent was evaporated. The residue waspurified by flash column chromatography (silica gel, 60:20 hexanes:ethylacetate), affording 119 mg (78%) of the intermediate hydroxy ketone.

The hydroxy-ketone was hydrolyzed to the carboxylic acid according tothe procedure outlined in Example 1. The corresponding hydroxy-acid (117mg, 0.52 mmol) was dissolved in diethyl ether (5 mL) at 0° C., and asolution of diazomethane in diethyl ether (5 mL) was added followed byboron trifluoride diethyl etherate (3 drops). A white precipitate formedand nitrogen gas was evolved. After 30 min, the mixture was filtered andthe filtrate was concentrated. Purification of the residue by flashcolumn chromatography on silica gel (70:30 hexanes:ethyl acetate) gavethe 4-methoxy-methyl ester as a colorless oil (67 mg, 51%). The methylester was carried forward according to the procedure described inExamples 71b through 71d to furnish the title compound as a white solid(27 mg, 30%). R_(f)=0.34 in (10:90 methanol:chloroform); ¹H-NMR (400MHz, CDCl₃) δ 7.32 (d, J=15.5 Hz, 1H), 6.20 (d, J=15.5 Hz, 1H), 4.63 (s,2H), 3.82-3.40 (m, 9H), 3.38 (d, J=11.50 Hz, 3H), 2.43 (dd, J=17.5, 5.5Hz, 1H), 2.03 (dd, J=17.5, 9.5 Hz, 1H), 1.83 (d, J=13.5 Hz, 1H), 1.76(s, 3H), 1.40 (t, J=12.0 Hz, 1H), 1.09 (s, 3H), 1.08 (s, 3H); Massspectrum (ESI+ve) m/z 336 (MH⁺).

Example 94 (−)-((1R,6S)-2,2,6-trimethylcyclohexyl)methyl4-carbamoylpiperazine-1-carboxylate

Sodium borohydride (78.0 mg, 2.07 mmol) was added to a stirred solutionof (1S,6S)-2,2,6-trimethylcyclohexanecarbaldehyde (320 mg, 2.07 mmol) inmethanol (10 mL) at 0° C. under argon. The reaction was stirred at roomtemperature for 16 hours. The reaction was quenched by adding water (60mL) and extracted with diethyl ether (4×25 mL). The combined organiclayers were dried over sodium sulfate, filtered and concentrated. Theintermediate alcohol was purified by flash column chromatography (0-25%ethyl acetate in hexanes) and obtained as a clear oil (248 mg, 77%).

The alcohol (235 mg, 1.51 mmol) was dissolved in anhydrousdichloromethane (20 mL) and cooled to 0° C. To this stirred solution wasadded triphosgene (179 mg, 0.61 mmol) and pyridine (227 mg, 2.88 mmol).The reaction was stirred at 0° C. until complete consumption of thestarting alcohol by TLC. tert-Butyl piperazine-1-carboxylate (338 mg,1.81 mmol) was then added to the reaction mixture in one portion. Thereaction was stirred at room temperature for 18 hours. The reactionmixture was then diluted with dichloromethane (30 mL) and extracted with1M HCl (2×15 mL) and saturated NaHCO₃ solution (15 mL). The organiclayer was washed with brine (20 mL) then dried over sodium sulfate,filtered and concentrated. The intermediate was purified by flash columnchromatography (0-25% ethyl acetate in hexanes) and obtained as a clearoil (394 mg, 70%).

The Boc-protected intermediate was carried forward following theprocedure of Example 71d and was substituted for tert-butyl4-((E)-3-((1,6-anti)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxylate (71c). The title compound, obtained as a whitesolid (188 mg, 88%). [α]_(D) ²³=−0.4° (c=0.25, chloroform); R_(f)=0.45in (10:90 methanol:chloroform); Mp=127-130° C.; ¹H-NMR (400 MHz,DMSO-d₆) δ 6.05 (br s, 2H), 4.17 (dd, J=11.5, 3.5 Hz, 1H), 4.01 (dd,J=11.5, 3.5 Hz, 1H), 3.30 (dd, J=18.5, 13.5 Hz, 8H), 1.68-1.59 (m, 1H),1.53 (m, 1H), 1.42 (m, 2H), 1.31 (d, J=13.0 Hz, 1H), 1.25-1.12 (m, 1H),0.95 (m, 2H), 0.87 (d, J=6.5 Hz, 3H), 0.82 (s, 6H); Mass spectrum(ESI+ve) m/z 312 (MH⁺).

Example 95 (−)-N¹-Methyl-N¹-(((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)piperazine-1,4-dicarboxamide

Sodium triacetoxyborohydride (4.10 g, 19.5 mmol) was added to a stirredsolution of (1S,6S)-2,2,6-trimethylcyclohexanecarbaldehyde (1.00 g, 6.48mmol) and methyl amine hydrochloride (1.30 g, 19.5 mmol) in a 10:1 (v/v)mixture of DMF (13 mL) and acetic acid (1.3 mmol). The reaction wasstirred at room temperature for 16 hours. The reaction was quenched byadding a saturated solution of sodium carbonate (20 mL) and diluted withdistilled water (60 mL). The aqueous solution was extracted with diethylether (3×75 mL). The combined organic layers were washed with brine (100mL), dried over magnesium sulfate, filtered and concentrated to give aclear oil (678 mg).

The amine (253 mg, 1.50 mmol) was dissolved in anhydrous dichloromethane(20 mL) and cooled to 0° C. To this stirred solution was addedtriphosgene (179 mg, 0.61 mmol) and pyridine (227 mg, 2.88 mmol). Thereaction was stirred at 0° C. until complete consumption of the startingamine by TLC. tert-Butyl piperazine-1-carboxylate (338 mg, 1.81 mmol)was then added to the reaction mixture in one portion. The reaction wasstirred at room temperature for 18 hours. The reaction mixture was thendiluted with dichloromethane (30 mL) and extracted with 1M HCl (2×15 mL)and saturated NaHCO₃ solution (15 mL). The organic layer was washed withbrine (20 mL) then dried over sodium sulfate, filtered and concentrated.The intermediate was purified by flash column chromatography (0-50%ethyl acetate in hexanes) and obtained as a clear oil (400 mg, 70%).

The Boc-protected intermediate was carried forward following theprocedure of Example 71d and was substituted for tert-butyl4-((E)-3-((1,6-ante)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxylate (71c). The title compound, obtained as a whitesolid (87.3 mg, 92%). [α]_(D) ²³=−7.2° (c=0.25, chloroform); R_(f)=0.35in (10:90 methanol:chloroform); Mp=152-157° C.; ¹H-NMR (400 MHz,DMSO-d₆) δ 6.03 (br s, 2H), 3.32-3.19 (m, 5H), 3.09-3.03 (m, 3H),3.00-2.93 (m, 2H), 2.80 (d, J=11.5 Hz, 3H), 1.56 (d, J=12.5 Hz, 1H),1.39 (m, 2H), 1.35-1.26 (m, 2H), 1.17 (m, 1H), 0.99-0.89 (m, 5H), 0.82(d, J=6.5 Hz, 3H), 0.77 (s, 3H); Mass spectrum (ESI+ve) m/z 325 (MH⁺).

Example 96N¹-(((1R,6S)-2,2,6-trimethylcyclohexyl)methyl)piperazine-1,4-dicarboxamide

Hydroxylamine hydrochloride (1.35 g, 19.5 mmol) was dissolved in a 1:1(v/v) solution of ethanol and water, and treated with sodium bicarbonate(1.64 g, 19.5 mmol) at room temperature for 10 minutes.(1S,6S)-2,2,6-trimethylcyclohexanecarbaldehyde (1.00 g, 6.48 mmol) wasadded and the reaction mixture was heated to reflux and stirred for 3days. The reaction was concentrated and the residue dissolved in brine(50 mL) and extracted with chloroform (3×25 mL). The combined organiclayers were dried over magnesium sulfate, filtered and concentrated togive the oxime as a clear oil (˜1.1 g).

The crude oxime (750 mg, 4.43 mmol) was dissolved in anhydroustetrahydrofuran (5 mL) and cooled to 0° C. To this stirred solution wasadded lithium aluminum hydride (168 mg, 4.43 mmol), and the reaction washeated to reflux for 18 hours. The reaction slurry was filtered througha pad of Celite and washed with tetrahydrofuran (10 mL). The crudemixture was then concentrated and the residue dissolved in diethyl ether(5 mL). The solution was filtered through a plug of silica gel andeluted with 10% methanol in chloroform (3×50 mL). The solution was thenconcentrated to give the product amine as a colorless oil (200 mg, 29%).

The amine (200 mg, 1.29 mmol) was dissolved in anhydrous dichloromethane(20 mL) and cooled to 0° C. To this stirred solution was addedtriphosgene (573 mg, 1.93 mmol) and triethylamine (522 mg, 5.16 mmol).The reaction was stirred at 0° C. until complete consumption of thestarting amine by TLC. tert-Butyl piperazine-1-carboxylate (264 mg, 1.42mmol) was then added to the reaction mixture in one portion. Thereaction was stirred at room temperature for 18 hours. The reaction wasquenched with a saturated solution of ammonium chloride (30 mL)extracted with dichloromethane (3×20 mL). The combined organic layerswere washed with brine (20 mL) then dried over magnesium sulfate,filtered and concentrated. The intermediate was purified by flash columnchromatography (20-40% ethyl acetate in hexanes) and obtained as a whitesolid (360 mg, 76%).

The Boc-protected intermediate was carried forward following theprocedure of Example 71d and was substituted for tert-butyl4-((E)-3-((1,6-anti)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxylate (71c). The title compound, obtained as a yellowsolid (63.0 mg, 74%). R_(f)=0.30 in (10:90 methanol:chloroform); Mp=158°C. (decomp); ¹H-NMR (400 MHz, CD₃OD) δ 3.43 (m, 8H), 3.13 (m, 1H), 1.65(s, 1H), 1.54-1.33 (m, 4H), 1.32-1.23 (m, 1H), 1.05-1.00 (m, 5H), 0.97(d, J=6.5 Hz, 3H), 0.87 (s, 3H); Mass spectrum (ESI+ve) m/z 311 (MH⁺).

Example 97 4-(((1R,6S)-2,2,6-trimethylcyclohexanecarboxamido)methyl)piperidine-1-carboxamide

(1 S,6S)-2,2,6-trimethylcyclohexanecarbaldehyde (2.75 g, 17.8 mmol) wasadded dropwise to a solution of 60% nitric acid (1.5 mL) at 55° C. andstirred for 30 minutes. The reaction was then cooled to room temperatureand diluted with water (10 mL) and neutralized with sodium bicarbonate.The aqueous solution was then extracted with dichloromethane (3×5 mL).The aqueous layers was then acidified with 1M HCl until pH=1.0, andextracted with diethyl ether (3×10 mL). The combined organic layers weredried over magnesium sulfate, dried and concentrated to obtain the crudeacid as a solid (2.00 g, 66%).

To a solution of crude acid (170 mg, 1.00 mmol) in acetonitrile (3.0 mL)was added 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 418 mg, 1.10 mmol). The solution was stirredat room temperature for 30 minutes then diisopropylethylamine (383 μL,2.20 mmol) and tert-butyl 4-(aminomethyl)piperidine-1-carboxylate (214mg, 1.00 mmol) was added to the reaction mixture. The reaction was thenstirred at room temperature for 16 hours. The reaction was quenched witha 1M solution of hydrochloric acid (2 mL) and the biphasic mixture wasseparated. The organic layer was concentrated in vacuo (40° C.) and thecrude material loaded on to silica gel for purification via flash columnchromatography running an isocratic eluent of 30% ethyl acetate inhexanes. The title compound was isolated as a white solid (160 mg, 30%).

The Boc-protected intermediate was carried forward following theprocedure of Example 71d and was substituted for tert-butyl4-((E)-3-((1,6-anti)-2,2,6-trimethylcyclohexyl)acryloyl)piperazine-1-carboxylate (71c). The title compound, obtained as a whitesolid (22.0 mg, 24%). R_(f)=0.22 in (10:90 methanol:chloroform);Mp=185-187° C.; ¹H-NMR (400 MHz, CDCl₃) δ 5.61 (br s, 1H), 4.57 (br s,2H), 3.94 (d, J=12.5 Hz, 2H), 3.21 (td, J=12.5, 6.5 Hz, 1H), 3.14-3.03(td, J=12.5, 6.5 Hz, 1H), 2.79 (t, J=12.5 Hz, 2H), 1.86 (m, 1H), 1.71(m, 4H), 1.55-1.44 (m, 2H), 1.38 (m, 2H), 1.28-1.06 (m, 4H), 1.00 (s,3H), 0.92 (s, 3H), 0.83 (m, 4H); Mass spectrum (ESI+ve) m/z 310 (MH⁺).

Example 98(E)-2-(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)azetidin-3-yl)acetamide 98a (E)-Methyl2-(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)azetidin-3-yl)acetate

The title compound, obtained as a yellow oil (650 mg, 85%), was preparedfrom the product of Example 71b by following the procedure of Example 3except methyl 2-(azetidin-3-yl)acetate was substituted for methylaminehydrochloride. R_(f)=0.29 in (60:40 ethyl acetate:hexanes); ¹H-NMR (400MHz, DMSO-d₆) δ 7.76 (d, J=15.5 Hz, 1H), 6.42 (d, J=15.5 Hz, 1H), 4.92(t, J=8.5 Hz, 1H), 4.78-4.66 (m, 1H), 4.48 (dd, J=8.5, 5.5 Hz, 1H), 4.25(dd, J=10.5, 5.5 Hz, 1H), 4.17 (s, 3H), 3.52 (td, J=13.5, 5.5 Hz, 1H),3.23 (d, J=7.5 Hz, 2H), 2.57 (t, J=6.0 Hz, 2H), 2.26 (s, 3H), 2.14 (td,J=8.5, 6.0 Hz, 2H), 1.99 (dd, J=7.5, 4.0 Hz, 2H), 1.56 (s, 6H); Massspectrum (ESI+ve) m/z 306 (MH⁺).

98b(E)-2-(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)azetidin-3-yl)acetamide

(E)-Methyl2-(1-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)azetidin-3-yl)acetate (100 mg, 0.344 mmol) was dissolved in a solution of 7N ammoniain methanol (2.0 mL) and stirred at room temperature for 2 days. Thetitle compound was purified by preparative plate thin layerchromatography (5:95 methanol:chloroform) to afford a clear oil (45.5mg, 48%). R_(f)=0.60 in (10:90 methanol:chloroform); ¹H-NMR (400 MHz,CDCl₃) δ 7.28 (d, J=15.5 Hz, 1H), 5.81 (d, J=15.5 Hz, 1H), 5.79 (br s,1H), 5.52 (br s, 1H), 4.39 (t, J=8.5 Hz, 1H), 4.23 (t, J=9.5 Hz, 1H),3.90 (dd, J=8.0, 5.5 Hz, 1H), 3.74 (dd, J=10.0, 5.5 Hz, 1H), 3.13-2.91(m, 1H), 2.67-2.47 (m, 2H), 2.03 (t, J=6.0 Hz, 2H), 1.72 (s, 3H), 1.59(dd, J=12.0, 6.90 Hz, 2H), 1.50-1.41 (m, 2H), 1.03 (s, 6H); Massspectrum (ESI+ve) m/z 291 (MH⁺).

Example 99 (E)-3-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acrylamido)azetidine-1-carboxamide 99a (E)-tert-Butyl3-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamido)azetidine-1-carboxylate

The title compound, obtained as a clear oil (172 mg, 97%), was preparedfrom the product of Example 1 by following the procedure of Example 3except tert-butyl 3-aminoazetidine-1-carboxylate was substituted formethylamine hydrochloride. R_(f)=0.40 in (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, DMSO-d₆) δ 7.10 (s, 1H), 6.49 (d, J=16.0 Hz, 1H), 5.13(d, J=16.0 Hz, 1H), 3.80 (m, 1H), 3.42 (m, 2H), 3.01 (m, 2H), 1.28 (t,J=6.0 Hz, 2H), 0.95 (s, 3H), 0.89-0.80 (m, 2H), 0.70 (m, 2H), 0.63 (s,9H), 0.26 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 349 (MH⁺).

99b(E)-3-(3-(2,6,6-Trimethylcyclohex-1-en-1-yl)acrylamido)azetidine-1-carboxamide

The title compound, obtained as a white solid (17 mg, 12%), was preparedfrom the product of Example 99a by following the procedure of Example71d. Mp=185-186° C. (decomp); R_(f)=0.30 in (10:90methanol:dichloromethane); ¹H-NMR (400 MHz, MeOD) δ7.91 (s, 1H), 7.30(d, J=16.0 Hz, 1H), 5.96 (d, J=16.0 Hz, 1H), 4.70-4.59 (m, 1H), 4.25 (t,J=8.0 Hz, 2H), 3.84 (dd, J=8.5, 5.50 Hz, 2H), 2.08 (t, J=6.0 Hz, 2H),1.77 (s, 3H), 1.66 (m, 2H), 1.51 (m, 2H), 1.07 (s, 6H) ppm; Massspectrum (ESI+ve) m/z 292 (MH⁺).

Example 100(E)-3-(2,6,6-Trimethylcyclohex-1-en-1-yl)-N-(2-ureidoethyl)acrylamide100a (E)-tert-Butyl (2-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamido) ethyl)carbamate

The title compound, obtained as a white solid (262 mg, 77%), wasprepared from the product of Example 1 by following the procedure ofExample 3 except tert-butyl (2-aminoethyl)carbamate was substituted formethylamine hydrochloride. R_(f)=0.34 in (50:50 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 7.28 (d, J=15.5 Hz, 1H), 6.29 (br s, 1H), 5.74(d, J=15.5 Hz, 1H), 5.01 (br s, 1H), 3.45 (dd, J=11.0, 5.5 Hz, 2H), 3.32(d, J=5.5 Hz, 2H), 2.02 (t, J=7.0 Hz, 2H), 1.71 (s, 3H), 1.65-1.56 (m,2H), 1.49-1.38 (m, 11H), 1.03 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z337 (MH⁺).

100b(E)-3-(2,6,6-Trimethylcyclohex-1-en-1-yl)-N-(2-ureidoethyl)acrylamide

The title compound, obtained as a white solid (40.0 mg, 24%), wasprepared from the product of Example 100a by following the procedure ofExample 71d. Mp=165-167° C.; R_(f)=0.30 in (10:90methanol:dichloromethane); ¹H-NMR (400 MHz, MeOD) δ 7.29 (d, J=16.0 Hz,1H), 5.95 (d, J=16.0 Hz, 1H), 3.44-3.18 (m, 4H), 2.10 (t, J=6.0 Hz, 2H),1.78 (s, 3H), 1.73-1.61 (m, 2H), 1.53 (m, 2H), 1.09 (s, 6H) ppm; Massspectrum (ESI+ve) m/z 280 (MH⁺).

100c(E)-N-(2-(2,2,2-Trifluoroacetamido)ethyl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamide

The title compound, obtained as a white solid (45.0 mg, 23%), wasisolated as a by-product from the above reaction: Example 100b.R_(f)=0.50 in (10:90 methanol:chloroform); ¹H-NMR (400 MHz, MeOD) δ 7.29(d, J=16.0 Hz, 1H), 5.93 (d, J=16.0 Hz, 1H), 3.46 (s, 4H), 2.10 (t,J=6.0 Hz, 2H), 1.77 (s, 3H), 1.67 (m, 2H), 1.52 (m, 2H), 1.08 (s, 6H)ppm; ¹⁹F-NMR (376 MHz, CDCl₃) δ −77.4 (s) ppm; Mass spectrum (ESI+ve)m/z 333 (MH⁺).

Example 101(E)-N-Methyl-N-(2-(1-methylureido)ethyl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamide 101a(E)-N-Methyl-N-(2-(methylamino)ethyl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamide

The title compound, obtained as a white solid (200 mg, 75%), wasprepared from the product of Example 1 by following the procedure ofExample 3 except N¹,N²-dimethylethane-1,2-diamine (10 equiv) wassubstituted for methylamine hydrochloride. R_(f)=0.20 in (10:90methanol:dichloromethane); ¹H-NMR (400 MHz, CDCl₃) δ 7.31 (d, J=15.5 Hz,1H), 6.21 (d, J=15.5 Hz, 1H), 3.74 (m, 2H), 3.36 (m, 2H), 3.16 (s, 3H),2.86 (s, 3H), 2.05 (m, 2H), 1.76 (s, 3H), 1.67-1.56 (m, 2H), 1.51-1.42(m, 2H), 1.05 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 265 (MH⁺).

101b(E)-N-Methyl-N-(2-(1-methylureido)ethyl)-3-(2,6,6-trimethylcyclohex-1-en-1-yl)acrylamide

The title compound, obtained as a white solid (36.0 mg, 26%), wasprepared from the product of Example 101a by following the procedure ofExample 71d. Mp=118-120° C.; R_(f)=0.18 in (5:95methanol:dichloromethane); ¹H-NMR (400 MHz, CDCl₃) δ 7.33 (d, J=15.5 Hz,1H), 6.20 (d, J=15.5 Hz, 1H), 5.01 (br s, 2H), 3.55 (m, 2H), 3.47-3.38(m, 2H), 3.13 (s, 3H), 2.96 (s, 3H), 2.04 (t, J=6.0 Hz, 2H), 1.75 (s,3H), 1.62 (m, 2H), 1.50-1.43 (m, 2H), 1.05 (s, 6H) ppm; Mass spectrum(ESI+ve) m/z 308 (MH⁺).

Example 102(E)-4-(3-(2,2,6,6-Tetramethylcyclohexyl)acryloyl)piperazine-1-carboxamide102a (E)-Ethyl 3-(2,2,6,6-tetramethylcyclohexyl)acrylate

The title compound, obtained as a colorless oil (0.901 g, 80%), wasprepared by following the procedure of Example 71a except2,2,6,6-tetramethylcyclohexanecarbaldehyde was substituted for(1,6-anti)-2,2,6-trimethylcyclohexanecarbaldehyde. R_(f)=0.66 (5:95ethyl acetate:hexanes); ¹H NMR (400 MHz, CDCl₃) δ 6.96 (dd, J=15.5, 11.0Hz, 1H), 5.77 (d, J=15.5 Hz, 1H), 4.19 (q, J=7.0 Hz, 2H), 1.68-1.56 (m,2H), 1.50-1.43 (m, 3H), 1.30 (t, J=7.0 Hz, 3H), 1.19-1.10 (m, 2H), 0.97(s, 6H), 0.79 (m, 6H) ppm; Mass spectrum (ESI+ve) m/z 239 (MH⁺).

102b (E)-3-(2,2,6,6-Tetramethylcyclohexyl)acrylic acid

The title compound, obtained as a white solid (0.720 g, 93%), wasprepared from the product of 102a by following the procedure of Example60c. Mp=138-140° C.; R_(f)=0.20 (10:90 ethyl acetate:hexanes); ¹H NMR(400 MHz, CDCl₃) δ 7.10 (dd, J=15.5, 11.0 Hz, 1H), 5.80 (d, J=15.5 Hz,1H), 1.70 (d, J=11.0 Hz, 1H), 1.59 (m, 1H), 1.47 (m, 3H), 1.21-1.11 (m,2H), 0.98 (s, 6H), 0.80 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 211(MH⁺).

102c (E)-tert-Butyl4-(3-(2,2,6,6-tetramethylcyclohexyl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a white solid (210 mg, 88%), wasprepared from the product of Example 102b by following the procedure ofExample 3 except tert-butyl piperazine-1-carboxylate was substituted formethylamine hydrochloride. R_(f)=0.40 in (25:75 ethyl acetate:hexanes);¹H-NMR (400 MHz, CDCl₃) δ 6.91 (dd, J=15.0, 11.0 Hz, 1H), 6.15 (d,J=15.0 Hz, 1H), 3.73-3.37 (m, 8H), 1.62 (m, 2H), 1.52-1.36 (m, 11H),1.21-1.06 (m, 2H), 0.97 (s, 6H), 0.79 (s, 6H) ppm; Mass spectrum(ESI+ve) m/z 379 (MH⁺).

102d(E)-4-(3-(2,2,6,6-tetramethylcyclohexyl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a white solid (111 mg, 74%), wasprepared from the product of example 102c by following the procedure ofExample 71d. Mp=172-174° C.; R^(f)=0.34 in (10:90 methanol:chloroform);¹H-NMR (400 MHz, DMSO-d₆) δ 6.65 (dd, J=15.0, 11.0 Hz, 1H), 6.42 (d,J=15.0 Hz, 1H), 6.04 (s, 2H), 3.48 (m, 4H), 3.35-3.26 (m, 4H), 1.73 (d,J=11.0 Hz, 1H), 1.57 (d, J=13.0 Hz, 1H), 1.42 (m, 3H), 1.14 (m, 2H),0.91 (s, 6H), 0.76 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 322 (MH⁺).

Example 103(E)-1-Morpholino-3-(2,2,6,6-tetramethylcyclohexyl)prop-2-en-1-one

The title compound, obtained as a white solid (42.1 mg, 51%), wasprepared from the product of Example 102b by following the procedure ofExample 3 except morpholine was substituted for methylaminehydrochloride. Mp=84-85° C.; R_(f)=0.35 in (30:70 ethylacetate:hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 6.92 (dd, J=15.0, 11.0 Hz,1H), 6.13 (d, J=15.0 Hz, 1H), 3.63 (m, 8H), 1.63 (m, 2H), 1.51-1.40 (m,3H), 1.15 (m, 2H), 0.97 (s, 6H), 0.79 (s, 6H) ppm; Mass spectrum(ESI+ve) m/z 280 (MH⁺).

Example 104N-((2,6,6-Trimethylcyclohex-1-en-1-yl)methyl)morpholine-4-carboxamide

The title compound, obtained as a white solid (337 mg, 77%), wasprepared from 2-(2,6,6-trimethylcyclohex-1-en-1-yl)acetic acid byfollowing the procedure of Example 55a except morpholine was substitutedfor tert-butyl piperazine-1-carboxylate. Mp=89-91° C.; R_(f)=0.20 in(25:75 ethyl acetate: hexanes); ¹H-NMR (400 MHz, CDCl₃) δ 3.99 (br s,1H), 3.79 (d, J=3.5 Hz, 2H), 3.69-3.62 (m, 4H), 3.32-3.25 (m, 4H), 1.93(t, J=6.0 Hz, 2H), 1.64 (s, 3H), 1.60-1.53 (m, 3H), 1.45-1.37 (m, 2H),0.98 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 267 (MH⁺).

Example 105 (E)-4-(3-(2,6,6-Trimethycyclohex-1-en-1-yl)acryloyl)piperazine-1-carbothioamide

The product of Example 28b (865 mg, 2.39 mmol) was dissolved indichloromethane (18 mL) was added dropwise a 4.0 M solution ofhydrochloric acid in 1,4-dioxane (6.0 mL, 23.9 mmol). The reactionmixture was stirred at room temperature for 18 hours then concentratedin vacuo.

The crude material was dissolved in dichloromethane (100 mL) andextracted with 1M sodium hydroxide (3×50 mL) and then washed with brine(50 mL). The organic layer was dried over magnesium sulfate, filteredand concentrated in vacuo to give a crude oil.

The crude oil was dissolved in tetrahydrofuran (20 mL) andtriphenylmethylisothiocyanate (719 mg, 2.39 mmol) was added to thesolution. The reaction was heated to reflux for 7 days., and thenconcentrated to dryness. Purification via preparative plate thin layerchromatography (7:93 methanol:chloroform) afforded the title compound asa white solid (42.0 mg, 5%). R_(f)=0.35 (10:90methanol:dichloromethane); ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J=15.5 Hz,1H), 6.17 (d, J=15.5 Hz, 1H), 5.82 (s, 2H), 4.12 (s, 2H), 3.91-3.67 (m,6H), 2.04 (d, J=6.0 Hz, 2H), 1.75 (s, 3H), 1.66-1.59 (m, 2H), 1.52-1.42(m, 2H), 1.05 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 322 (MH⁺).

Example 106(E)-2-Ethynyl-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide106a. tert-Butyl 4-tritylpiperazine-1-carboxylate

A solution of Boc-piperazine (14.3 g, 77.0 mmol) and triethylamine (11.0mL, 77.0 mmol) in dichloromethane (300 mL) was stirred at roomtemperature under argon. To the reaction flask was added trityl chloride(21.5 g, 77.0 mmol) and the reaction mixture was stirred for 1 hour atroom temperature. The solution was then washed with saturated ammoniumchloride (100 mL), water (100 mL), dried over sodium sulfate, filteredand concentrated in vacuo. The title compound was isolated as a whitesolid (11.8 g, 36%). R_(f)=0.86 (20:80 ethyl acetate:hexane); ¹H NMR(400 MHz, CDCl₃) δ 7.49 (m, 6H), 7.34-7.24 (m, 6H), 7.18 (m, 3H),3.63-3.49 (m, 4H), 2.57-1.93 (m, 4H), 1.42 (s, 9H) ppm; Mass spectrum(ESI+ve) m/z 329 (MH⁺-tBu).

106b. tert-Butyl 2-formyl-4-tritylpiperazine-1-carboxylate

A solution of the product of Example 106a (7.30 g, 170 mmol) andN¹,N¹,N²,N²-tetramethylethylene-1,2-diamine (3.90 mL, 26.0 mmol) inanhydrous diethyl ether (250 mL) was stirred at −78° C. under argon. Tothis stirred solution was added sec-butyllithium (1.4 M in cyclohexane,18.5 mL, 26.0 mmol) over 10 minutes. The reaction mixture was stirredfor 1 hour at −78° C., then DMF (2.00 mL, 26.0 mmol) was added in oneportion and the mixture was stirred for 1 hour at −78° C.

The reaction was quenched by the addition of saturated ammonium chloride(30 mL) at −78° C. The solution was vigorously stirred and allowed towarm to room temperature over 40 minutes. The reaction mixture wasconcentrated in vacuo, and the residue diluted with brine (60 mL) andextracted with chloroform (3×100 mL). The combined organic phases weredried over sodium sulfate, filtered and concentrated. The title compoundwas isolated as a white solid (7.42 g, 96%). ¹H NMR displays a mixtureof rotamers. R_(f)=0.33 (10:90 ethyl acetate:hexane); ¹H NMR (400 MHz,CDCl₃) δ 9.85 (m, 2H), 7.45 (m, 12H), 7.36-7.25 (m, 12H), 7.20 (m, 6H),4.60 (s, 1H), 4.41 (s, 1H), 3.93-3.71 (m, 2H), 3.69-3.32 (m, 4H), 2.98(m, 2H), 1.98 (s, 2H), 1.56-1.35 (m, 22H) ppm; Mass spectrum (ESI+ve)m/z 401 (MH⁺-tBu).

Example 106c tert-Butyl 2-ethynyl-4-tritylpiperazine-1-carboxylate

The Ohira-Bestmann reagent was prepared in situ by stirring dimethyl(2-oxopropyl) phosphonate (0.720 mL, 5.30 mmol),4-acetamidobenzenesulfonyl azide (1.30 g, 5.30 mmol) and potassiumcarbonate (1.82 g, 13.0 mmol) in acetonitrile (60 mL) at roomtemperature for 18 hours. A slurry of the product of Example 106b (2.00g, 4.40 mmol) in methanol (12 mL) was added and the mixture was stirredat room temperature for 18 hours.

The reaction mixture was concentrated in vacuo, and the residue wastaken up in ethyl acetate (150 mL) and washed with brine (100 mL). Theaqueous phase was extracted with ethyl acetate (2×100 mL). The combinedorganic phases were washed with saturated sodium bicarbonate (150 mL),brine (100 mL), dried over sodium sulfate, filtered and concentrated invacuo.

The product was purified by column chromatography (neutral alumina,isocratic elution 40:60 dichloromethane:hexane). The title compound wasisolated as a white foam (0.80 g, 40%). R_(f)=0.57 (20:80 ethylacetate:hexane); ¹H NMR (400 MHz, CDCl₃) δ 7.90-7.39 (m, 6H), 7.30 (m,6H), 7.17 (m, 3H), 4.98-4.68 (m, 1H), 3.80-3.73 (m, 1H), 3.63-3.41 (m,1H), 3.35 (m, 1H), 3.17-2.99 (m, 1H), 2.54 (s, 1H), 1.75-1.64 (m, 1H),1.42 (s, 9H) ppm; Mass spectrum (ESI+ve) m/z 211 (MH⁺-trityl).

Example 106d tert-Butyl 2-ethynylpiperazine-1-carboxylate

To a solution of the product of Example 106c (0.350 g, 0.800 mmol) indichloromethane (5.0 mL) at 0° C. was added a trichloroacetic acid (2%w/v in dichloromethane, 5.0 mL). The mixture was stirred at 0° C. for 25minutes then quenched with aqueous sodium hydroxide (1N, 10 mL). Theorganic phase was removed and the aqueous phase was extracted withdichloromethane (3×10 mL). The combined organic phases were dried oversodium sulfate, filtered and concentrated in vacuo. The product waspurified by column chromatography (40 g silica gel, gradient elution5:95:0.1 methanol:dichloromethane:ammonium hydroxide to 10:90:0.1methanol:dichloromethane:ammonium hydroxide). This provided the titlecompound as clear oil (48 mg, 29%). R_(f)=0.5 (10:90 ethylacetate:hexane); Mass spectrum (ESI+ve) m/z 211 (MH⁺).

Example 106e (E)-tert-Butyl2-ethynyl-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxylate

The title compound, obtained as a white solid (60 mg, 68%), was preparedfrom the product of Example 1 by following the procedure of Example 3except the product of Example 106d was substituted for methylaminehydrochloride. R_(f)=0.32 (30:70 ethyl acetate:hexane); ¹H NMR (400 MHz,CDCl₃) 7.38 (d, J=15.5 Hz, 1H), 6.24 (d, J=15.5 Hz, 1H), 5.13-4.60 (m,2H), 3.90 (m, 2H), 3.21 (m, 2H), 2.26 (s, 1H), 2.05 (t, J=6.0 Hz, 2H),1.75 (s, 3H), 1.62 (m, 2H), 1.35-1.13 (m, 2H), 1.06 (m, 6H) ppm; Massspectrum (ESI+ve) m/z 387 (MH⁺).

Example 106f(E)-2-Ethynyl-4-(3-(2,6,6-trimethylcyclohex-1-en-1-yl)acryloyl)piperazine-1-carboxamide

The title compound, obtained as a clear oil (12 mg, 23%), was preparedfrom the product of Example 106e by following the procedure of Example71d. The ¹H NMR exhibits a mixture of rotamers. R_(f)=0.20 in (10:90methanol:chloroform); ¹H-NMR (400 MHz, CDCl₃) δ 7.41-7.31 (m, 1H),6.29-6.13 (m, 1H), 5.13-4.99 (m, 1H), 4.87 (s, 2H), 4.78-4.64 (m, 1H),4.16-4.01 (m, 1H), 3.60-3.46 (m, 1H), 3.38 (s, 2H), 2.86-2.73 (m, 1H),2.32-2.26 (m, 1H), 2.06-2.00 (m, 2H), 1.73 (s, 3H), 1.60 (d, J=5.54 Hz,2H), 1.49-1.42 (m, 2H), 1.21 (m, 3H), 1.04 (m, 6H) ppm; Mass spectrum(ESI+ve) m/z 330 (MH⁺).

Example 107(E)-1-morpholino-3-(3,3,6,6-tetramethylcyclohex-1-enyl)prop-2-en-1-oneExample 107a 1,4,4-trimethylcyclohex-2-enol

The title compound, obtained as a light yellow oil (41 g, 90%), wasprepared from 4,4-dimethylcyclohex-2-enone (40 g, 0.3 mol) according tothe procedure of [Dauben, W.; Michno, D. J. Org. Chem. 1977, 42,682-685]. R_(f)=0.5 (5:1 petroleum ether:ethyl acetate) ¹H NMR (400 MHz,CDCl₃) δ 5.46 (d, J=10.0 Hz, 1H), 5.43 (d, J=10.0 Hz, 1H), 1.73-1.70 (m,2H), 1.59-1.56 (m, 1H), 1.50-1.45 (m, 1H), 1.27 (s, 3H), 1.01 (s, 3H),0.95 (s, 3H) ppm; Mass spectrum (ESI+ve) m/z 123 (MH-H₂O)⁺.

Example 107b 3,6,6-trimethylcyclohex-2-enone

The title compound, obtained as a colorless oil (14 g, 35%), wasprepared from the product of Example 107a (40 g, 0.3 mol) according tothe procedure of [Dauben, W.; Michno, D. J. Org. Chem. 1977, 42,682-685]. R_(f)=0.4 (5:1 petroleum ether:ethyl acetate) ¹H NMR (400 MHz,CDCl₃) δ 5.77 (s, 1H), 2.29 (t, J=6.0 Hz, 2H), 1.93 (s, 3H), 1.80 (d,J=6.0 Hz, 3H), 1.09 (s, 6H) ppm; Mass spectrum (ESI+ve) m/z 139 (MH)⁺.

Example 107c 2,2,5,5-Tetramethylcyclohexanone

CuI (6.9 g, 36.2 mmol) was added to a dry 250-mL round-bottom flaskequipped with a stir bar and sealed under argon with a septum. The flaskwas evacuated with a vacuum pump and purged with argon. This process wasrepeated three times. THF (75 mL) was injected and the slurry was cooledto −78° C., where MeLi (45 mL, 72 mmol) was added dropwise. The mixturewas allowed to warm until homogeneous and was recooled to −78° C., whereBF₃.Et₂O (8.9 mL, 72 mmol) was added via a syringe. The product ofExample 107b (5.0 g, 36.2 mmol) was added neat and the reaction mixturewas stirred for 1.5 h. The reaction was quenched with 250 mL of a 10%NH₄OH/90% saturated NH₄Cl solution and then extracted with ethyl acetate(250 mL), the organic layer was washed with aqueous saturated sodiumbicarbonate (50 mL×2), brine (50 mL), dried over sodium sulfate andconcentrated to give a colorless oil (3.5 g) which was a mixture of theproduct and starting material. The mixture was chromatographed to affordthe title compound as a colorless solid (1.5 g 26%).

¹H NMR (400 MHz, CDCl₃) δ 2.21 (s, 2H), 1.69-1.65 (m, 2H), 1.61-1.57 (m,2H), 1.09 (s, 6H), 0.94 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 216.36,51.32, 44.00, 36.89, 36.62, 34.69, 28.5, 25.15; Mass spectrum (ESI+ve)m/z 155 (MH)⁺.

Example 107d Methyl3-(1-hydroxy-2,2,5,5-tetramethylcyclohexyl)propiolate

To a solution of lithium diisopropyl amide (3.1 mL, 2M in diethyl ether,6.2 mmol) in THF (5 mL), cooled to −78° C., was added methyl propiolate(520 mg, 6.2 mmol) in THF (1 mL) dropwise. The mixture was stirred atthis temperature for 1 h, and a solution the product of Example 107c(420 mg, 3.0 mmol) in THF (2 mL) was added dropwise. The mixture wasstirred at −78° C. for 1 h. The mixture was quenched with aqueousammonium chloride (10 mL), extracted with ethyl acetate (50 mL), washedwith sodium bicarbonate (10 mL), brine (10 mL), dried over sodiumsulfate, and concentrated. The residue was purified by columnchromatography (10:1 petroleum ether:ethyl acetate) to give the titlecompound as a light yellow oil (600 mg, yield: 40%). ¹H NMR (400 MHz,CDCl₃) δ 3.79 (s, 3H), 1.92 (s, 1H), 1.81 (d, J=14.3 Hz, 1H), 1.70 (d,J=14.3 Hz, 1H), 1.66-1.62 (m, 1H), 1.43-1.31 (m, 3H), 1.12 (s, 3H),1.055 (s, 3H), 1.050 (s, 3H), 1.02 (s, 3H); Mass spectrum (ESI+ve) m/z221 (MH-H₂O)⁺.

Example 107e (E)-methyl3-(1-hydroxy-2,2,5,5-tetramethylcyclohexyl)acrylate

The product of Example 107d (589 mg, 2.47 mmol) in THF (10.0 mL) wasadded to a solution of Red-Al (4.95 mmol, 3.5 M in toluene, 1.4 mL) inTHF (8 mL) dropwise at −72° C. (dry ice-ethanol bath) under nitrogenatmosphere. After stirring at the same temperature for 30 min, themixture was quenched with 0.1 M HCl (5 mL), extracted with ethyl acetate(50 mL), washed with 0.1 M HCl (20 mL×3), aqueous sodium bicarbonate (20mL) and brine (20 mL), dried over sodium sulfate and concentrated underreduced pressure. The resulting residue was purified by columnchromatography (10:1 petroleum ether:ethyl acetate) to give the desiredproduct as white solid (380 mg, yield: 64%). ¹H NMR (400 MHz, DMSO-d₆) δ7.10 (d, J=15.6 Hz, 1H), 6.07 (d, J=15.6 Hz, 1H), 3.76 (s, 3H), 1.88(td, J=13.6, 4.0 Hz, 1H), 1.73 (d, J=14.6 Hz, 1H), 1.48 (td, J=13.6, 4.0Hz, 1H), 1.34 (td, J=4.0, 2.0 Hz, 1H), 1.31 (s, 1H), 1.25 (dd, J=14.6,1.9 Hz, 1H), 1.17 (dt, J=13.6, 4.0 Hz, 1H), 1.11 (s, 3H), 0.98 (s, 3H),0.94 (s, 3H), 0.89 (s, 3H); Mass spectrum (ESI+ve) m/z 241 (MH)⁺.

Example 107f (E)-methyl 3-(3,3,6,6-tetramethylcyclohex-1-enyl)acrylate

To a solution of the compound of Example 107e (380 mg, 1.58 mmol) inacetic acid (1.8 mL) was added acetic anhydride (0.6 mL) followed byacetyl chloride (0.6 mL). The mixture was refluxed for 2 h. The reactionsolution was concentrated. The residue was taken up in ethyl acetate (50mL), washed with aqueous sodium bicarbonate (30 mL×3) and brine (30 mL),dried over sodium sulfate and concentrated. The crude product waspurified by column chromatography (10:1 petroleum ether:ethyl acetate)to give the title compound as light yellow oil (230 mg, yield: 65%). ¹HNMR (400 MHz, MeOD) δ 7.33 (d, J=15.8 Hz, 1H), 6.03 (d, J=15.8 Hz, 1H),5.78 (s, 1H), 3.76 (s, 3H), 1.57-1.51 (m, 2H), 1.51-1.45 (m, 2H), 1.11(s, 6H), 1.02 (s, 6H); Mass spectrum (ESI+ve) m/z 223 (MH)⁺.

Example 107g (E)-3-(3,3,6,6-tetramethylcyclohex-1-enyl)acrylic acid

To a solution of the compound of Example 107f (150 mg, 0.67 mmol) inmethanol (5 mL) and water (1 mL) was added sodium hydroxide (80 mg, 2.0mmol). The mixture was refluxed for 2 h. The reaction solution wasconcentrated, acidified to ph˜2-3, extracted with ethyl acetate (50 mL),dried over sodium sulfate, and concentrated. The residue was dried undervacuum to give the title compound as colorless oil (130 mg, yield: 93%).The crude compound was used in the next step without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ 7.42 (d, J=15.8 Hz, 1H), 6.04(d, J=15.8 Hz, 1H), 5.86 (s, 1H), 1.58-1.52 (m, 2H), 1.52-1.45 (m, 2H),1.12 (s, 6H), 1.04 (s, 6H).

Example 107h(E)-1-morpholino-3-(3,3,6,6-tetramethylcyclohex-1-enyl)prop-2-en-1-one

A solution of the compound of Example 107g (60 mg, 0.29 mmol), HATU (165mg, 0.435 mmol), diisopropylethyl amine (112 mg, 0.87 mmol) in DMF (2mL) was stirred at rt for 0.5 h. Morpholine (25 mg, 0.29 mmol) wasadded. The mixture was stirred at rt for 3 h. After dilution with water(10 mL), the mixture was extracted with ethyl acetate (50 mL), washedwith water (20 mL×2), brine (20 mL×2), dried sodium sulfate, andconcentrated. The residue was purified by column chromatography (2:1petroleum ether; ethyl acetate) to give the title compound as acolorless syrup (65 mg, yield: 81%). Preparative HPLC gave 30 mg of thetitle product as a white solid (30 mg). Further purification byprep-HPLC gave 8 mg of the pure title compound as light yellow solid, ¹HNMR (400 MHz, CDCl₃) δ 7.36 (d, J=15.1 Hz, 1H), 6.41 (d, J=15.1 Hz, 1H),5.71 (s, 1H), 3.79-3.54 (m, 8H), 1.57-1.51 (m, 2H), 1.51-1.45 (m, 2H),1.10 (s, 6H), 1.03 (s, 6H); Mass spectrum (ESI+ve) m/z 278 (MH)⁺.

Example 108 (E)-4-(3-(3,3,6,6-tetramethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide Example 108a Tert-Butyl4-carbamoylpiperazine-1-carboxylate

To a solution of tert-butyl piperazine-1-carboxylate (5.0 g, 26.8 mmol)in acetic acid (15 mL) and water (25 mL) was added a solution ofpotassium cyanate (11.25 g, 138.9 mmol) in water (25 mL) dropwise. Afteraddition the mixture was stirred at rt for 4 h, during which time asolid precipitated. The solid was collected by filtration, re-dissolvedin dichloromethane (20 mL), dried oversodium sulfate, and filtered. Thefiltrate was concentrated to give the title compound as a white solid(3.3 g, yield: 53%), which was used in the next step without furtherpurification. ¹H NMR (400 MHz, DMSO-d₆) δ 6.04 (s, 2H), 3.26 (s, 8H),1.41 (s, 9H).

Example 108b Piperazine-1-carboxamide trifluoroacetate salt

A solution of the product of Example 108a (1.5 g, 6.5 mmol) intrifluoroacetic acid (5 mL) and dichloromethane (15 mL) was stirred atrt for 3 h. The mixture was concentrated. The residue was trituratedwith ethyl acetate (5 mL×2) and diethyl ether (5 mL×2), dried undervacuum to give the title compound as colorless syrup (1.5 g, yield:95%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.11 (s, 2H), 7.04-5.66 (br, s, 2H),3.57-3.45 (m, 4H), 3.06 (s, 4H).

Example 108c (E)-4-(3-(3,3,6,6-tetramethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

A solution of the product of Example 107g (65 mg, 0.31 mmol), HATU (178mg, 0.47 mmol), diisopropylethyl amine (120 mg, 0.93 mmol) in DMF (2 mL)was stirred at rt for 0.5 h. The product of Example 108b (75 mg, 0.31mmol) was added. The mixture was stirred at rt for 3 h. After dilutionwith water (10 mL), the mixture was extracted with ethyl acetate (50mL), washed with water (20 mL×2), brine (20 mL×2), driedover sodiumsulfate, and concentrated. The residue was purified by columnchromatography (10:1 dichloromethane:methanol) to afford the titlecompound as a white solid (70 mg, yield: 70%). Further purification byprep-HPLC gave a white solid (23 mg, 23%). ¹H NMR (400 MHz, DMSO-d₆) δ7.37 (d, J=15.0 Hz, 1H), 6.42 (d, J=14.8 Hz, 1H), 5.74 (s, 1H), 4.59 (s,2H), 3.90-3.32 (m, 8H), 1.58-1.52 (m, 2H), 1.52-1.46 (m, 2H), 1.10 (s,6H), 1.04 (s, 6H); Mass spectrum (ESI+ve) m/z 320 (MH)⁺.

Example 109(E)-4-(3-(3,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamideExample 109a 2,5,5-Trimethylcyclohexanone

To a solution of the product of Example 107b (1.0 g, 7.24 mmol) inmethanol (20 mL) was added Pd/C (0.2 g). The mixture was stirred at 25°C. under a hydrogen atmosphere overnight. The reaction mixture wasfiltered through Celite and the filtrate was concentrated under reducedpressure to give a colorless oil which was purified by chromatography togive the title compound as colorless oil (300 mg, 30%). R_(f)=0.6 (5:1petroleum ether:ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 5.77 (s, 1H),2.29 (t, J=6.0 Hz, 2H), 1.93 (s, 3H), 1.80 (d, J=6.0 Hz, 3H), 1.09 (s,6H); ¹³CNMR (100 MHz, CDCl₃) δ 215.49, 46.21, 44.09, 39.53, 34.72,29.70, 25.02, 24.95, 21.89; Mass spectrum (ESI+ve) m/z 141 (MH)⁺.

Example 109b Methyl 3-(1-hydroxy-2,2,5-trimethylcyclohexyl)propiolate

To a solution of methyl propiolate (0.6 g, 7.13 mmol) in THF (12 mL),cooled to −78° C., was added dropwise lithium diisopropyl amide solution(3.6 mL, 2M in ether, 7.13 mmol). The mixture was stirred at thistemperature for 1 h, and a solution of the product of Example 109a (1.0g, 7.13 mmol) in THF (12 mL) was added dropwise. The mixture was stirredat −78° C. for 1 h. The mixture was quenched with ammonium chloride (aq.5 mL), extracted with ethyl acetate (30 mL), washed with sodiumbicarbonate (aq. 10 mL), brine (10 mL), dried over sodium sulfate andconcentrated. The residue was purified by column chromatography (10:1petroleum ether:ethyl acetate) to give a light yellow oil (0.76 g, 48%).¹H NMR (400 MHz, CDCl₃) δ 3.80 (s, 3H), 2.04 (s, 1H), 1.86-1.77 (m, 2H),1.66-1.57 (m, 2H), 1.55-1.48 (m, 1H), 1.44-1.37 (m, 1H), 1.28 (t, J=7.1Hz, 1H), 1.16-1.12 (s, 3H), 0.99 (s, 3H), 0.97 (d, J=6.3 Hz, 3H).

Example 109c (E)-methyl 3-(I-hydroxy-2,2,6-trimethylcyclohexyl)acrylate

To a solution of Red-Al (1.9 mL, 6.7 mmol) in THF (11 mL) under argon,cooled to −72° C.; was added dropwise the product of Example 109b (0.75g, 3.35 mmol) in THF (14 mL). The mixture was stirred at thistemperature for 1 h. The mixture was quenched with 0.1 M HCl (150 mL).The solution was concentrated under reduced pressure and was thendiluted with ethyl acetate (60 mL). The mixture was separated and theaqueous layer was extracted with ethyl acetate (60 mL). The combinedorganic layers were then washed with sat. sodium bicarbonate (15 mL),dried over sodium sulfate and concentrated under reduced pressure. Theresulting residue was purified by column chromatography (10:1 petroleumether; ethyl acetate) to give the title compound as light yellow oil(0.55 g, 73%). ¹H NMR (400 MHz, CDCl₃) δ 7.45 (d, J=15.5 Hz, 1H), 6.17(d, J=15.5 Hz, 1H), 3.77 (s, 3H), 1.68 (m, 1H), 1.65 (s, 2H), 1.60 (d,J=1.9 Hz, 1H), 1.57 (t, J=3.4 Hz, 1H), 1.51 (d, J=3.0 Hz, 1H), 1.46 (dd,J=4.2, 2.1 Hz, 1H), 1.43 (d, J=3.9 Hz, 1H), 1.28-1.19 (m, 3H), 1.03 (s,3H), 0.94 (d, J=7.9 Hz, 3H), 0.85 (s, 3H); Mass spectrum (ESI+ve) m/z209 (MH-H₂O)⁺.

Example 109d (E)-methyl 3-(3,6,6-trimethylcyclohex-1-enyl)acrylate

To a solution of the product of Example 109c (120 mg, 0.53 mmol) incarbon tetrachloride (5 mL) at 0° C. was added a solution of Martin'ssulfurane (0.9 g, 1.33 mmol) in carbon tetrachloride (7.5 mL) underargon. After addition, the cooling bath was removed and the mixture wasstirred at rt for 1.5 h. Crushed ice and water (15 mL) were added andafter being stirred for 20 min, the mixture was extracted withdichloromethane (50 mL). The organic phases were washed with water (5mL) and brine (5 mL), dried over sodium sulfate and concentrated todryness. The residue was purified by preparative thin layerchromatography to afford the title compound as colorless oil (75 mg,68%). ¹H NMR (400 MHz, CDCl₃) δ 7.22 (d, J=15.8 Hz, 1H), 6.00 (d, J=15.8Hz, 1H), 5.89 (d, J=2.9 Hz, 1H), 3.70 (s, 3H), 2.22 (m, 1H), 1.76-1.65(m, 11H), 1.61 (s, 1H), 1.58-1.46 (m, 1H), 1.49-1.37 (m, 1H), 1.30-1.17(m, 1H), 1.07 (d, J=2.7 Hz, 6H), 0.98 (d, J=6.7 Hz, 3H).

Example 109e (E)-3-(3,6,6-trimethylcyclohex-1-enyl)acrylic acid

To a solution of the product of Example 109d (200 mg, 0.96 mmol) inmethanol (7 mL) and water (1 mL) was added sodium hydroxide (115 mg,2.88 mmol). The mixture was refluxed for 2 h. The reaction mixture wasconcentrated under reduced pressure and then it was diluted with water(5 mL). 3N HCl was added to adjust the pH to 2. The aqueous layer wasextracted with ethyl acetate (3×10 mL), washed with brine (5 mL), driedover sodium sulfate and concentrated under reduced pressure to give thecrude title product as a brown oil. (170 mg) which was carried onwithout further purification. ¹H NMR (400 MHz, DMSO) δ 12.25 (s, 1H),7.16 (d, J=15.9 Hz, 1H), 6.05-5.93 (m, 2H), 2.25 (d, J=7.3 Hz, 1H),1.75-1.65 (m, 1H), 1.57-1.51 (m, 1H), 1.47-1.38 (m, 1H), 1.26-1.18 (m,1H), 1.06 (d, J=6.4 Hz, 6H), 0.98 (d, J=7.2 Hz, 3H); Mass spectrum(ESI+ve) m/z 195 (MH)⁺.

Example 109f(E)-4-(3-(3,6,6-trimethylcyclohex-1-enyl)acryloyl)piperazine-1-carboxamide

To a solution of the product of Example 109e (85 mg, 0.44 mmol) and HATU(250 mg, 0.66 mmol) in DMF (3 mL) was added the product of Example 108b(107 mg, 0.44 mmol) followed by diisopropylethyl amine (171 mg, 1.32mmol). The mixture was stirred at rt for 2 h. Water (20 mL) was added tothe reaction mixture and it was extracted with ethyl acetate (3×10 mL).The organic layer was washed with brine (5 mL), dried over sodiumsulfate and concentrated under reduced pressure. The obtained residuewas purified by preperative thin layer chromatography to give acolorless syrup. Further purification by column chromatography (20:1dichloromethane:methanol) gave the title compound as a white solid. (36mg, 27%). ¹H NMR (400 MHz, CDCl₃) δ 7.38 (d, J=15.2 Hz, 1H), 6.44 (d,J=15.1 Hz, 1H), 5.88 (d, J=2.8 Hz, 1H), 4.60 (s, 2H), 3.71 (d, J=26.5Hz, 4H), 3.52 (s, 4H), 2.26 (d, J=7.5 Hz, 1H), 1.82-1.68 (m, 1H), 1.58(ddd, J=13.1, 6.0, 3.0 Hz, 1H), 1.54-1.44 (m, 1H), 1.34-1.20 (m, 1H),1.11 (d, J=5.1 Hz, 6H), 1.05 (d, J=7.1 Hz, 3H); Mass spectrum (ESI+ve)m/z 306 (MH)⁺.

Example 110(E)-1-morpholino-3-(3,6,6-trimethylcyclohex-1-enyl)prop-2-en-1-one

To a solution of the product of Example 109e (90 mg, 0.46 mmol) and HATU(266 mg, 0.7 mmol) in DMF (2 mL) was added morpholine (40 mg, 0.46 mmol)followed by diisopropylethyl amine (178 mg, 1.38 mmol). The mixture wasstirred at rt for 2 h. Water (20 mL) was added to the reaction mixtureand it was extracted with ethyl acetate (3×10 mL). The organic layer waswashed with brine (5 mL), dried over sodium sulfate and concentratedunder reduced pressure. The obtained residue was purified by preparativethin layer chromatography and then column chromatography (2:1 petroleumether; ethyl acetate) to give the semi pure title compound as a lightyellow oil (85 mg). Preparative HPLC gave 36 mg of the desired productas colorless syrup (36 mg). Further purification by columnchromatography (2:1 petroleum ether:ethyl acetate) gave the titlecompound (10 mg, 8%)¹H NMR (400 MHz, CDCl₃) δ 7.37 (d, J=15.2 Hz, 1H),6.43 (d, J=15.1 Hz, 1H), 5.86 (d, J=2.8 Hz, 1H), 3.68 (d, J=34.3 Hz,8H), 2.25 (d, J=7.1 Hz, 1H), 1.74 (ddd, J=13.2, 6.3, 3.0 Hz, 1H),1.62-1.40 (m, 1H), 1.33-1.16 (m, 2H), 1.10 (t, J=6.7 Hz, 6H), 1.03 (d,J=7.1 Hz, 3H); Mass spectrum (ESI+ve) m/z 264 (MH)⁺.

Biology Examples

In carrying out the procedures of the present invention it is of courseto be understood that reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

The invention is described in more detail in the following non-limitingexamples. It is to be understood that these particular methods andexamples in no way limit the invention to the embodiments describedherein and that other embodiments and uses will no doubt suggestthemselves to those skilled in the art.

Reagents

Monoclonal anti-rhodopsin 1D4 antibody can be purchased from Universityof British Columbia.

Cell Lines and Culture Conditions

Stable cell lines expressing opsin protein were generated using theFlp-In T-Rex system. The stable cells were grown in DMEM high glucosemedia supplemented with 10% (v/v) fetal bovine serum,antibiotic/antimycotic solution, 5 μ/ml blasticidin and hygromycin at37° C. in presence of 5% CO₂. For all the experiments the cells wereallowed to reach confluence and were induced to produce opsin with 1μg/ml tetracycline after change of media and then compounds were added.The plates were incubated for 48 hours after which the cells wereharvested.

SDS-PAGE and Western Blotting

Proteins were separated on SDS-PAGE gels and western blotted asdescribed in (Noorwez et al., J. Biol. Chem. 279, 16278-16284 (2004)).

The in vivo efficacy of the compounds of the invention in treatingmacular degeneration can be demonstrated by various tests well known inthe art. For example, human patients are selected based on a diagnosisof macular degeneration (such as where there is a gross diagnosis ofthis condition or where they have been shown to exhibit build-up oftoxic visual cycle products, such as A2E, lipofuscin, or drusen in theireyes. A compound of the invention, such as that of Formula I and/orFormula II, is administered to a test group while a placebo, such as PBSor DMSO, is administered to a control group that may be as large or maybe somewhat smaller than the test group. The test compound isadministered either on a one time basis or on a sequential basis (forexample, weekly or daily) or according to some other predetereminedschedule

Administration of the test compound is normally by oral or parenteralmeans and in an amount effective to retard the development and/orreoccurrence of macular degeneration. An effective dose amount isgenerally in the range of about 1 to 5,000 mg or in the range of 10 to2,000 mg/kg. Administration may include multiple doses per day.

Efficacy of the test compound in retarding progression of maculardegeneration is generally by measuring increase in visual acuity (forexample, using Early Treatment Diabetic RP Study (ETDRS) charts(Lighthouse, Long Island, N.Y.). Other means of following and evaluatingefficacy is by measuring/monitoring the autofluorescence or absorptionspectra of such indicators as N-retinylidene-phosphatidylethanolamine,dihydro-N-retinylidene-N-retinyl-phosphatidylethanolamine,N-retinylidene-N-retinyl-phosphatidylethanolamine,dihydro-N-retinylidene-N-retinyl-ethanolamine, and/orN-retinylidene-phosphatidylethanolamine in the eye of the patient.Autofluorescence is monitored using different types of instrument, forexample, a confocal scanning laser ophthalmoscope.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) is acommon pathological feature observed in various degenerative diseases ofthe retina. A toxic vitamin A-based fluorophore (A2E) present withinlipofuscin granules has been implicated in death of RPE andphotoreceptor cells. Such experiments can employ an animal model whichmanifests accelerated lipofuscin accumulation to evaluate the efficacyof a therapeutic approach based upon reduction of serum vitamin A(retinol). Administration of test compound to mice harboring a nullmutation in the Stargardt's disease gene (ABCA4) produces reductions inserum retinol/retinol binding protein and arrested accumulation of A2Eand lipofuscin autofluorescence in the RPE.

Test animals are available for use in testing efficacy of a testcompound in reducing build-up of toxic pigments, such as lipofuscin. Forexample, mice have been produced that exhibit increased production ofsich toxic product. Such mice have been described in the literature(see, for example, Widder et al., U.S. Pub. 2006/0167088) and theirvalue and utility are well known to those in the art.

Showing the efficacy of compounds of the invention in protecting againstlight toxicity is conveniently performed by methods well known in theart (see, for example, Sieving et al, PNAS, Vol. 98, pp 1835-40 (2001)).

Biology Example 1 SDS-PAGE and Western Blotting

Proteins were separated on SDS-PAGE gels and western blotted asdescribed in (Noorwez et al., J. Biol. Chem. 279, 16278-16284 (2004)).HEK-P23H cell opsin expression was induced as described above for 16 to24 hrs in the presence of DMSO (blank) or various concentrations of testcompound (generally 1 to 40 μM). After incubation cells were lysed incold Phosphate-Buffered Saline with 1% docecyl maltoside (PBD-D) for 1hr, and the lysate cleared by centrifugation. Total protein (≈10 μg) wasloaded on 4-20% SDS polyacrylamide-gels (BioRad) and total opsinquantified by western blotting using the anti-rhodopsin monoclonalantibody 1D4 (2.5 μg/mL) as the primary antibody and IRDye-labeled goatanti-mouse (Licor) as the secondary antibody for detection. The blotswere scanned and opsin levels quantified using the Odyssey infraredscanner and software (Licor Biosystems).

Here, a test compound is added to a selected final concentration (20 μMresults are reported in Table 1). The results were calculated as the %of mature P23H opsin (˜52 kDA) produced in the HEK 293 cells relative tothe control 9-cis retinal at 20 uM defined as producing a 100% response

TABLE 1 Activity in the western blot assay: Compound No. Increase inMature P23H Opsin 6 113% @ 20 μM 14 144% @ 20 μM 15 113% @ 20 μM 17 132%@ 20 μM 21  89% @ 20 μM 29  97% @ 20 μM 34 155% @ 20 μM 37 136% @ 20 μM44 136% @ 20 μM 45 122% @ 20 μM

The western blot results show the total amount of opsin protein produced(as quantified on the gel). A 52 kDA band is the fully maturatedprotein. Rhodopsin generation data then allows determination as towhether it is suitably folded to form rhodopsin when exposed to retinal.Data has shown that not all mature protein is necessarily folded toaccept retinal and form pigment but the mutant protein in the presenceof chaperone does appear to traffic normally out of the edoplasmicreticulum.

Biology Example 2 Rhodopsin Purification and Regeneration

P23H cell opsin expression is induced 48 hrs in the presence of DMSO(blank) or various concentrations of test compound (generally 1 to 40μM). P23H opsin producing cells are washed with PBS and lysed in coldPBS-D for 1 hour. The lysate is cleared by centrifugation and added to1D4-coupled sepharose beads and incubated for 1 hour at 4° C. Opsin waseluted from the antibody beads with a competing peptide corresponding tothe last 18 amino acids of rhodopsin in the same buffer. The purifiedopsin is immediately used for rhodopsin regeneration studies using 9-cisretinal as chromophore. Opsin (≈25 M) is mixed with 10 μM 9-cis retinaland the absorbance is determined over the range of 250-650 nm every twominutes in a Cary 50 spectrophotometer (Varian) until no more rhodopsinis regenerated as measured by the increase in 480-500 nm absorbance.FIGS. 2-17 are the spectral results using selected compounds accordingto Biology Example 2.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A compound having the structure of Formula IA-B-Q-V   Formula I wherein A is:

B is —(CH₂)_(n)—, —CH═CH—, —CH₂—N(R²²)—, —CH₂—O—, —C(O)—CH₂—C(O)—,

or —C(O)NR²²—, wherein n=0, 1 or 2; E is —N(R²²)— or oxygen; Q is—C(O)—, —(CH₂)_(a)—, —S(O₂)— or —CH₂—C(O)—, wherein a is 1 or 2; V isNR²¹R²²,

wherein b is 1 or 2 and a is 1 or 2; Y is NR²², N-Q-U, CR²²R²³, oxygen,S(O)_(n), N—C(S)—NR²²R²³, N—(C═N—CN)—NR²²R²³, N—(C═N—SO₂CH₃)—NR²²R²³,C═NOR²², C═N—NR²²R²³ or CH-Q-U, n is 0, 1 or 2; U is NR²²R²³, loweralkyl, haloalkyl, alkoxy, OR²² or hydrogen; X is hydrogen, alkyl, or—C≡CR⁹; R¹ and R² are independently —CH₃ or —CH₂CH₃; R³ is hydrogen,—CH₃ or —CH₂CH₃; R_(a) and R_(b), are each independently hydrogen,deuterium or —CH₃—; R_(c) and R_(d), are each independently hydrogen,alkoxy, lower alkyl or alkenyl; R⁴ is —CH₃, —CF₃, —C₂H₅ or —C₃H₅; R⁵, R⁶and R⁷ are each independently hydrogen, lower alkyl, halogen,dialkylamine, nitro or dialkylamine; Z is CR³, CH or nitrogen; R⁸ is—CH₂— or —C(O)—; R⁹, R¹⁴ and R¹⁶ are each independently hydrogen, or—CH₃; R¹⁰ is N—R¹³, sulfur or oxygen; R¹¹ is ═N—, or ═C(CH₃)—; R¹² islower alkyl, alkoxy or haloalkyl; R¹³ is phenyl, lower alkyl orhaloalkyl; R¹⁵ is hydrogen or —C(O)CH₃; R¹⁷ and R¹⁸ together are—(CH₂)₄— or —CH═CH—CH═CH—; R19 and R20 together are—CH2-C(CH3)2-CH2-C(O)— or —CH═CH—CH═CH—; R²¹ is hydrogen, —C(O)CH₃, —CH₃or —CH₂CH₃; R²² and R²³ are each independently hydrogen or lower alkyl;R²⁴ and R²⁵ are each independently hydrogen or —CH₃; R²⁶ is NR²²R²³ oralkoxy; or wherein R¹ and R² taken together or R_(a) and R_(b) takentogether along with the carbon to which they are attached arecyclopropyl; or R²⁴ and R²⁵ taken together along with the two carbons towhich they are attached are cyclopropyl; or R²⁴ and R²⁵ taken togetheris oxo; T is oxygen, —N(R¹⁶)— or sulfur; and E is oxygen, —N(R¹⁶)—,sulfur or —C(O)—; including pharmaceutically acceptable salts, solvatesand hydrates thereof.
 2. The compound of claim 1, wherein A is


3. The compound of claim 2, wherein R_(a) and R_(b) are eachindependently hydrogen or methyl.
 4. The compound of claim 2, whereinR_(c) and R_(d) are each independently hydrogen or lower alkyl.
 5. Thecompound of claim 1, wherein V is:


6. The compound of claim 5, wherein a and b are each 1, X is hydrogen, Yis CH—C(O)NR²²R²³ or N—C(O)NR²²R²³ wherein R²³, R²⁴ and R²⁵ are allhydrogen.
 7. The compound of claim 1, wherein X is H, lower alkyl or—C≡CR⁹.
 8. The compound of claim 1, wherein Y is O or N—C(O)—NR²²R²³.9-20. (canceled)
 21. A method of reducing or inhibiting mislocalizationof an opsin protein, comprising contacting an opsin protein with acompound of claim
 1. 22. The method of claim 21, wherein said opsinprotein is pressent in a rod cell or a cone cell.
 23. (canceled)
 24. Themethod of claim 22, wherein said cell is present in a mammalian eye. 25.A method of inhibiting the formation or accumulation of a visual cycleproduct, comprising contacting an opsin protein with a compound ofclaim
 1. 26. The method of claim 25, wherein said visual cycle productis lipofuscin or N-retinylidene-N-retinylethanolamine (A2E).
 27. Amethod of treating or preventing an ophthalmic condition in a subject atrisk thereof, comprising administering to the subject an effectiveamount of a compound of claim
 1. 28. The method of claim 27, whereinsaid ophthalmic condition is an ocular protein mislocalization disorder.29. The method of claim 27, wherein said ophthalmic condition isselected from the group consisting of wet or dry age related maculardegeneration (ARMD), retinitis pigmentosa (RP), a retinal or maculardystrophy, Stargardt's disease, Sorsby's dystrophy, autosomal dominantdrusen, Best's dystrophy, peripherin mutation associate with maculardystrophy, dominant form of Stargart's disease, North Carolina maculardystrophy, light toxicity, normal vision loss related aging and normalloss of night vision related to aging.
 30. The method of claim 29,wherein said ophthalmic condition disorder is ARMD.
 31. The method ofclaim 29, wherein said ophthalmic condition is retinitis pigmentosa(RP).
 32. (canceled)
 33. A compound selected from the group consistingof:

or a pharmaceutically acceptable salt thereof.
 34. A compound having thestructure:

or a pharmaceutically acceptable salt thereof.